CN106754351A - External magnetic field auxiliary incubating device, method and its application - Google Patents
External magnetic field auxiliary incubating device, method and its application Download PDFInfo
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- CN106754351A CN106754351A CN201710151341.2A CN201710151341A CN106754351A CN 106754351 A CN106754351 A CN 106754351A CN 201710151341 A CN201710151341 A CN 201710151341A CN 106754351 A CN106754351 A CN 106754351A
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Abstract
The present invention relates to medical analysis instrument field, incubating device is aided in there is provided a kind of external magnetic field, including at least one set of magnet combination, the magnet combination is made up of discrete polylith cylindrical magnet, magnet combination described in every group is in clip-like, its jaws is shaped to cylindrical magnet described in polylith simultaneously near or against the container wall for holding thing to be incubated, and the magnetic pole of the same name of all cylindrical magnets is set towards the jaws.The invention provides a kind of external magnetic field incubating device and its application method, with magnetic field it is strong, apparatus structure is simple the characteristics of, be combined by by itself and motor and centrifugal device, improve integrated, the automaticity of equipment.
Description
Technical field
The present invention relates to full-automatic medical treatment detection device field, specifically provide one kind be used to help circulating tumor cell with
External magnetic field auxiliary incubating device, method and its application that antibody magnetic bead is combined.
Background technology
Circulating tumor cell (Circulating tumor cell, CTC) refers to come off from tumour and enter cyclic system
The cancer cell of system, it can be spontaneous or be come off from entity tumor focus (primary tumor or MET) by operation of diagnosis and treatment, is followed into body
Loop systems (such as peripheral blood).Most of CTC can occur apoptosis or swallowed after entering peripheral blood, and minority can escape and anchor disease
On tissue or organ beyond stove, and then develop into MET, increase malignant tumor patient mortality risk.These cells can be with
Found by simple blood testing, tumour, and it can be whether there is in indication body as the biological markers of cancer
Hereditary conditions and drug susceptibility.Different from surgical intervention, CTC is compatible with longitudinal research, being capable of effective guiding treatment.
CTC detections can be effectively applied to external early diagnosis, prognostic evaluation, (including rapid clinical chemotherapeutics is sieved individualized treatment
Choosing and the detection of drug resistance), tumor recurrence monitoring and the exploitation of new tumour medicine etc..But CTC detecting steps are more, it is numerous to operate
It is trivial, also counteracts that the popularization clinically of this technology.
Conventional CTC detections at present are divided into two steps, i.e. CTC enrichments (or capture) step and CTC detection and analysis steps.
Conventional CTC capture techniques have gradient density centrifugal technology, filter film technology, Flow Cytometry, microfluidic chip technology and exempt from
Epidemic disease magnetic bead technology.Wherein, immunomagnetic bead technique is related to the steps such as the mixing of sample, cell magnetic suck, is adapted in large volume loading
Under the conditions of a small amount of special cells are separated, for CTC detection application it is especially advantageous, its operating procedure is relatively easy, easily
Automated in realization.
At present the circulating tumor cell enrichment plant that has listed of the country be Johson ＆ Johnson cellsearch systems and
Ctcbiopsy systems (non-magnetic capture systems employ the mode of physical filtering).In order to antibody magnetic bead and CTC is better achieved
The combination of cell, generally carries out magnetic incubation using external magnetic field auxiliary incubating device to mixture.Cellsearch systems are based on
Immunological magnetic bead sorting primary is managed, and antibody magnetic bead is lifted to circulating tumor cell capture rate using outer magnet.Specifically utilize outer magnetic
Cycle back and forth movement of the body on guide rail, realizes the change in magnetic field in centrifuge tube, but its structure is more complicated, and enters to sample
During row absorption observation, it is found that the efficiency comparison that magnetic bead is combined with sample is low.Further, since being produced using outer magnet cycle back and forth movement
Magnetizing field change, it is necessary to be driven to it using extra motor, and is unfavorable for the miniaturization of equipment and integrated.
Therefore, those skilled in the art is devoted to developing a kind of simple structure, is easy to the external magnetic field of integration of equipments to incubate
Device is educated, incubation process is efficiently quickly and accurately completed.
The content of the invention
In view of the drawbacks described above of prior art, the technical problems to be solved by the invention are to provide a kind of external magnetic field auxiliary
Incubating device, including at least one set of magnet combination, the magnet combination are made up of discrete polylith cylindrical magnet, magnetic described in every group
Body combination is in clip-like, and its jaws is shaped to cylindrical magnet described in polylith simultaneously near or against the container device for holding thing to be incubated
Wall, the magnetic pole of the same name of all cylindrical magnets is set towards the jaws.
In a preferred embodiment of the invention, its incubating device also includes motor, centrifugal device, the driving
Motor is connected with the magnet combination, for driving the magnet combination near or against the centrifugal device.Its centrifugal device
Further use driving stepper motor.
In a preferred embodiment of the invention, the cylindrical magnet is neodymium iron boron magnetic body.
In a preferred embodiment of the invention, the height of the magnet combination is the container for holding thing to be incubated
1/3~2/3.The N-S directions of cylindrical magnet are axial direction, are normal direction perpendicular to N-S directions.By it due to holding thing to be incubated
Container is usually tube, and the magnetic field that magnet is provided needs to cover all liquid in pipe, it is therefore desirable to which magnet has in normal direction
There is certain height.
In a preferred embodiment of the invention, the quantity of the cylindrical magnet is 3.
Present invention also offers a kind of incubation method of external magnetic field auxiliary, comprise the following steps：
Magnet is near centrifuge tube；
It is incubated；
Stir and evenly mix；
Magnet leaves centrifuge tube；
Centrifuge rotates at least one station；
Repeat the above steps until incubation process is completed.
Present invention also offers application of the above-mentioned external magnetic field auxiliary incubating device in circulating tumor cell detecting system.
Invention further provides above-mentioned external magnetic field auxiliary incubation method answering in circulating tumor cell detecting system
With.
Technique effect
The invention provides a kind of external magnetic field incubating device and its application method, with magnetic field is strong, apparatus structure is simple
Feature, is combined by by it with motor and centrifugal device, improves integrated, the automaticity of equipment.
The technique effect of design of the invention, concrete structure and generation is described further below with reference to accompanying drawing, with
It is fully understood from the purpose of the present invention, feature and effect.
Brief description of the drawings
Fig. 1 is the structure front view of the magnet in a preferred embodiment of the present invention and centrifuge tube；
Fig. 2 is the structure top view of magnet and centrifuge tube in Fig. 1 embodiments；
Fig. 3 be Fig. 1 equivalent integers in magnet and centrifuge tube pair structure top view；
Fig. 4 is the structure top view that is installed in CTC tumour cell detecting systems of magnet combination of Fig. 1 embodiments.
Reference：1- neodymium iron boron magnetic bodies, 2- centrifuge tubes, 3- motors, 4- magnetic supports, 5- position-limit mechanisms.
Specific embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from start to finish
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
It is exemplary to scheme the embodiment of description, it is intended to for explaining the present invention, and be not considered as limiting the invention.
Embodiment 1
The external magnetic field auxiliary incubating device in a kind of circulating tumor cell detecting system for CTC is present embodiments provided,
As shown in figure 1, the funtion part construction unit of the incubating device is made up of one group of three magnet, the height h of three magnet is about
The 1/2 of test tube length, ensures that magnetic field has whole coverings of antibody magnetic bead part to whole test tube Nei Shengfa with this.
Three magnet is the clip-like combination of the N compositions extremely adjacent to each other of three pieces of column neodymium iron boron strong magnets 1, due to the same sex
The effect for repelling each other, three pieces of ndfeb magnets 1 are close to each other without contact, and the jaws shape for surrounding in detecting system according to using
The Size calculation of centrifuge tube 2 is obtained, as shown in Fig. 2 its jaws size fits into centrifuge tube 2 just, while keeping and centrifuge tube 2
It is in close contact, so as to keep the gravitation to the internal antibodies magnetic bead of centrifuge tube 2 so that antibody magnetic bead can be protected when close to three magnets
Hold displacement.
The construction unit is installed in CTC circulating tumor cell detecting systems, it is possible to use the combination with centrifugal device
The integration degree of raising equipment.As shown in figure 3,4 group of three magnet is installed on magnetic support 4, magnetic is driven by motor 3
Seat 4 is moved with three magnet, when motor 3 promotes magnetic support 4 to move to distalmost end, is approached against the lower end of centrifuge tube 2,
The high-intensity magnetic field that three magnet are produced, directed movement ability is improved the antibody magnetic bead in pipe under the influence of a magnetic field, captures whole blood
Analyte or extract in product.Then, motor 3 returns back to start bit, and the start bit position is limited by position-limit mechanism 5
Fixed, position-limit mechanism 5 limits the shift motion of magnetic support 4 with the male cooperation on track, and motor 3 can be coordinated to realize in work
Position and the rapid transfer of original position.The part coordinated with magnetic support 4 in centrifugal device is only gived in Fig. 3, in the present embodiment
Centrifugal platform can accommodate 12 centrifuge tubes 2, one group two-by-two, centrifugal platform uses driving stepper motor, therefore can be complete every time
Into after magnetic bead displacement, magnetic support 4 is recalled rapidly, while centrifugal mechanism rotates two stations, lower two groups of centrifuge tubes 2 is entered work
Make position, while by three magnet against to the tube wall of centrifuge tube 2.When centrifuge tube 2 is slowly moved with centrifugal pan, magnetic field disappears in pipe
Lose；After mixing is stirred to liquid in pipe, when centrifuge tube 2 is moved near the servicing unit again, realize same as described above
Function.The like, by magnetic bead directed movement for several times repeatedly, improve combination of the magnetic bead to analyte or extract general
Rate/capture rate.Whole process can automatically be quickly completed anti-by stepper motor and the translational speed of motor 3
The incubation process that body magnetic bead is combined with CTC cells.
External magnetic field auxiliary incubating device in use the present embodiment described below completes CTC cells and antibody magnetic bead was combined
The method of the incubation process of journey, method is comprised the following steps：
To contain to send out and need the correspondence position that the centrifuge tube for being incubated thing is put into centrifugal platform；
Enter position detection；
Motor drives three magnets near centrifuge tube to operating position, completes antibody magnetic bead displacement；
While keeping certain hour, mixing is stirred to the centrifuge tube of off-position using agitating device；
Motor drives three magnets to leave centrifuge tube to original position；
Centrifuge rotates two stations, other four centrifuge tubes is entered operating position；
Above step, certain time are repeated, until be entirely incubated process completing.
Embodiment 2
Device in embodiment 1 is used to improve antibody magnetic bead to analyte or the capture rate of extract, wherein described
Analyte or extract are circulating tumor cell.
By being confirmed after following verification experimental verification, the congenerous structure phase in device of the invention and cellsearch systems
Than, the capture rate of antibody magnetic bead can be greatly improved, the ability of circulating tumor cell is especially captured in whole blood sample, just
In carrying out further magnetic enrichment to it.
Test objective and design：The manual extraction CTC in whole blood matrix, contrasts different externally-applied magnetic fields to the CTC rate of recovery
Influence.
Experiment packet sets as follows：
Group 1：Using Johnson ＆ Johnson's reagent, the sample dilution buffer of mixing is added after whole blood addition human breast cancer cell SKBR3
(DB), magnetic bead, strengthens agent solution, and the magnetic field of magnets of incubation conditions three effect 2min, dropper is blown and beaten mixing 1min, stood repeatedly
2min, does 12 circulations, is extracted with double end bar magnet, sets diplopore experiment；
Group 2：After whole blood adds SKBR3 cells, Johnson ＆ Johnson DB, Johnson ＆ Johnson's magnetic bead and Genovo reinforcing agents that addition is mixed are incubated
Educate condition：Stirring rod is extracted in whole blood diverse location interval stirring 60min with bar magnet, sets diplopore experiment.
Group 3：Using Johnson ＆ Johnson's reagent, the DB of mixing, magnetic bead is added to strengthen agent solution, incubation conditions after whole blood addition SKBR3
Johnson ＆ Johnson magnetic field of magnets effect 2min, dropper blows and beats mixing 1min, stands 2min repeatedly, does 12 circulations, is carried out with double end bar magnet
Extract, diplopore experiment is set；
The test material for being used is referring to table 1 below.
The test material of table 1
Operating procedure includes：
1. prepare：2% bovine serum albumin(BSA) (BSA), room temperature closing are added to 96 orifice plates, 14mL round bottom centrifuge tubes used
10min, suction pipette head is blown and beaten 5 times using preceding in 2%BSA.
2. cell count：The BSA of 96 orifice plates of closing is exhausted and reclaimed, the SKBR3 that 50uL dilutes and dye is taken respectively thin
Born of the same parents, add into 96 orifice plates close, standing, with EVOS (a full-automatic micro imaging system of thermo companies production,
Automatically image scanning can be carried out to cell, and carries out semi-automatic counting) scan and count.
3. it is incubated：Added in the cell that will have been counted in the whole blood after cleaning, reagent is added according to the requirement of test group, and
Carry out incubation 1h.
4. adsorb：It is put into bar magnet to be adsorbed, condition：Bar magnet is four positions of whole blood (each displacement is 1.25cm)
It is each to stand 15 minutes, eutectoid content 1h.
5. clean：Bar magnet is transferred in the 24 hole depth orifice plates containing 5mL phosphate buffers (PBS) and is rinsed.
6. preserve：Bar magnet after cleaning is transferred in the 14mL round bottom centrifuge tubes containing 3mL PBS, waits to be counted.
Experimental result and discussion：
The each group comparison of test results of table 2
From Table 2, it can be seen that in the case of without outer magnet, capture rate is 79.7%；Using Johnson ＆ Johnson's outer magnet
When, capture rate is 88.7%；And when using outer magnet of the invention, capture rate is 103.5%.It follows that of the invention
Outer magnet can greatly improve capture rate.
Preferred embodiment of the invention described in detail above.It should be appreciated that one of ordinary skill in the art without
Need creative work just can make many modifications and variations with design of the invention.Therefore, all technologies in the art
Personnel are available by logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Technical scheme, all should be in the protection domain being defined in the patent claims.
Claims (9)
1. a kind of external magnetic field aids in incubating device, it is characterised in that including at least one set of magnet combination, the magnet combination is by dividing
Vertical polylith cylindrical magnet composition, magnet combination described in every group is in clip-like, and it is same that its jaws is shaped to cylindrical magnet described in polylith
When set towards the jaws near or against the container wall for holding thing to be incubated, the magnetic pole of the same name of all cylindrical magnets
Put.
2. external magnetic field as claimed in claim 1 aids in incubating device, it is characterised in that also including motor, centrifugal device,
The motor is connected with the magnet combination, for driving the magnet combination near or against the centrifugal device.
3. external magnetic field as claimed in claim 2 aids in incubating device, it is characterised in that the centrifugal device uses stepper motor
Drive.
4. external magnetic field as claimed in claim 1 aids in incubating device, it is characterised in that the cylindrical magnet is neodymium-iron-boron
Body.
5. external magnetic field as claimed in claim 1 aids in incubating device, it is characterised in that the height of the magnet combination is described
Hold the 1/3~2/3 of the container of thing to be incubated.
6. external magnetic field as claimed in claim 1 aids in incubating device, it is characterised in that column magnetic described in every group of magnet combination
The quantity of body is 3.
7. a kind of external magnetic field auxiliary incubation method, comprises the following steps：
Magnet is near centrifuge tube；
It is incubated；
Magnet leaves centrifuge tube；
Centrifuge rotates at least one station；
Stir and evenly mix；
Repeat the above steps until incubation process is completed.
8. the external magnetic field as described in claim 1-6 is any aids in incubating device answering in circulating tumor cell detecting system
With.
9. application of the external magnetic field auxiliary incubation method as claimed in claim 7 in circulating tumor cell detecting system.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710151341.2A CN106754351B (en) | 2017-03-14 | 2017-03-14 | External magnetic field assisted incubation device, method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710151341.2A CN106754351B (en) | 2017-03-14 | 2017-03-14 | External magnetic field assisted incubation device, method and application thereof |
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| CN106754351A true CN106754351A (en) | 2017-05-31 |
| CN106754351B CN106754351B (en) | 2023-07-25 |
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| CN102690786A (en) * | 2012-06-05 | 2012-09-26 | 武汉格蓝丽富科技有限公司 | Cell enriching, separating and extracting method and instrument and single cell analysis method |
| US20140242603A1 (en) * | 2013-02-26 | 2014-08-28 | Glenn Yaguang Deng | Methods and apparatus for target cell magnetic enrichment, isolation and biological analysis |
| CN104568923A (en) * | 2014-12-01 | 2015-04-29 | 浙江省肿瘤医院 | Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection |
| CN206591132U (en) * | 2017-03-14 | 2017-10-27 | 骏实生物科技(上海)有限公司 | External magnetic field aids in incubating device |
-
2017
- 2017-03-14 CN CN201710151341.2A patent/CN106754351B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481400A (en) * | 2009-01-07 | 2009-07-15 | 戴立忠 | Method for separating and purifying nucleic acid from sample of virus or cell containing nucleic acid and special magnetic separator therefor |
| CN102586225A (en) * | 2011-01-14 | 2012-07-18 | 博奥生物有限公司 | Method for separating target molecules by controlling magnetic beads |
| CN102690786A (en) * | 2012-06-05 | 2012-09-26 | 武汉格蓝丽富科技有限公司 | Cell enriching, separating and extracting method and instrument and single cell analysis method |
| US20140242603A1 (en) * | 2013-02-26 | 2014-08-28 | Glenn Yaguang Deng | Methods and apparatus for target cell magnetic enrichment, isolation and biological analysis |
| CN104568923A (en) * | 2014-12-01 | 2015-04-29 | 浙江省肿瘤医院 | Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection |
| CN206591132U (en) * | 2017-03-14 | 2017-10-27 | 骏实生物科技(上海)有限公司 | External magnetic field aids in incubating device |
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