CN103869060A - Circulating tumor stem cell detection kit based on magnetic beads and microfluidic chip - Google Patents
Circulating tumor stem cell detection kit based on magnetic beads and microfluidic chip Download PDFInfo
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Abstract
The invention provides a circulating tumor stem cell detection kit based on magnetic beads and a microfluidic chip. The circulating tumor stem cell detection kit is characterized by comprising a microfluidic chip, at least one immunomagnetic bead, and at least one fluorescent antibody against a tumor stem cell biomarker, wherein the immunomagnetic bead can be specifically bonded with the circulating tumor stem cell and marked with the tumor stem cell biomarker; the microfluidic chip comprises a glass chip base; a microfluidic channel is arranged on the glass chip base; a soft magnetic micro-array is arranged inside the microfluidic channel. By adopting the circulating tumor stem cell detection kit, a plurality of different categories of rare circulating tumor stem cells can be captured and detected by sampling once, and the circulating tumor stem cell detection kit has high sensitivity and specificity, is convenient and fast to operate, can easily collect captured cells, and does not need complicated surface modification processes of a first antibody and a second antibody of the microfluidic channel inside the chip.
Description
Technical field
The present invention relates to the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip.
Background technology
Hepatocellular carcinoma (Hepatocellular carcinoma, HCC) (abbreviation liver cancer) is one of modal malignant tumour in the world, liver cancer in the annual new diagnosis of China accounts for the whole world 55%, and its mortality ratio is row second in China's malignant tumour.Although excision is the preferred manner of current liver cancer treatment, even if radical excision still has the patient of 60-70% to occur transfer and relapse in 5 years.Liver cancer postoperative metastasis and recurrence are the postoperative major causes of death of patient, and it has become the main obstacle of further raising liver cancer treatment effect.Therefore, explore the mechanism of hepatoma Metastasis, recurrence, find more effective transfer early warning and curative effect evaluation means, to carry out early stage rationally intervention, become the key of further raising liver cancer survival rate.
Recently research prompting circulating tumor cell is played the part of pivotal player in metastases and recurrence.Enter in circulating but there is every day thousands of tumour cells to depart from primary tumor, but be not " seed " that each circulating tumor cell can become transfer and relapse.This is apart from outside environment (soil) factor, and " seed " self character is also determining that can it success " field planting " in new environment.We find that in early-stage Study in the quantity of the circulating tumor cell with stem cell characteristic and the early stage liver of Post hepatectomy of liver cancer, recurrence and lung transfer are closely related, for the circulating tumor cell compared with ripe, those circulating tumor stem cells have very strong one-tenth knurl, anti-apoptosis capacity.Therefore these cause " seed " cell of cancer metastasis, recurrence to have great clinical and significance of scientific research accurately to catch classification.But this group of cells are extremely rare in peripheral blood, average 10
7in individual leucocyte, only has a circulating tumor stem cell.All there is certain defect realizing catching aspect classification of circulating tumor stem cell as gradient density centrifugal technology, filter film technology and Flow Cytometry in current existing circulating tumor cell capture technique.Gradient density centrifugal and filter film technology are realized catching of cell by the difference (density, size) of physical characteristics between circulating tumor cell and haemocyte, because these two kinds its sensitivity of technology and the specificitys that realize catching based on physical characteristics are lower, cannot classify to catching cell, cell is easily lost, and has limited its application in circulating tumor cell is caught.Flow cytometry is by carrying out specific fluorescence labeling to testing sample, then utilize complicated Systems for optical inspection to identify sorting, though this technology can realize the categorised collection of different classes of tumour cell, but the cost costliness of equipment own, detection efficiency are low, need special messenger to operate, detect rare cell poor sensitivity simultaneously, cannot observe cytomorphology, also be difficult to be widely used in catching of circulating tumor stem cell.Therefore need exploitation badly a kind of cheap, simple to operate, disposable, there is the circulating tumor stem cell methods of high sensitivity, specificity and many marker detection.
Micro-fluidic chip technology has the plurality of advantages such as detection is efficient, integrated, reagent consumption is little and is just being applied to more and more biomedical sector, and has developed multiple micro-current controlled cell capture technique.But the cell capture chip that most has been reported still rests on catching of single mark, single kind cell, still lacks corresponding means for many marks, multi-class cell capture.Representative in them is that the one list mark circulating tumor cell of the reports on Nature in 2007 such as Nagrath S is caught chip, this chip arranges circular micro-pillar array in runner, through chemical modification, at runner inner wall and microtrabeculae surface conjunction epithelial cell adhesion factor antibody, in flowing by cell, carry out acquisition target cell with the collision of microtrabeculae.The success or not that this method is caught cell depends on whether target cell collides microtrabeculae, and therefore its capture rate is lower completely.Such chip cannot be realized the function that many marks circulating tumor stem cell catches by single injected sampling.In addition the application of this chip is also limited by the very difficult shortcoming such as collection and the chemical modification complexity of chip in early stage that discharges of the cell of catching.The object of catching in order to realize many marks cancer cell, Xu Y etc. proposes snake pipe for 2009 and delimit different capture regions on Anal Chem, in the upstream and downstream of each capture region, the perforate of turnover sample is set, the alternately open and closed system that utilizes perforate different aptamer separately on zones of different is fixing, reaches the object of parallel capture different carcinoma cell in same pipeline with this.Although the method can realize catching of many marks cell, but repeated runner fixedly makes, chip preparation process time is long, step is many, fixing owing to flowing in same pipeline again, easily produce the cross pollution of upstream and downstream, false positive is increased, and in order to control false positive rate, must lengthen the length in each region, this further integrated obstacle that caused to chip.
Summary of the invention
The object of this invention is to provide a kind of circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, without complicated chemical modification is carried out in the inside of its chip, possess sample feeding and just can comprise catching, identify and the function such as recovery of single, many marks circulating tumor stem cell to completing, also there is feature easy and simple to handle, integrated level is high simultaneously.
In order to achieve the above object, the invention provides a kind of circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, it is characterized in that, comprise micro-fluidic chip, at least one can with the immunomagnetic beads that is marked with tumor stem cell biomarker of circulating tumor stem cell specific binding and at least one fluorescence antibody for tumor stem cell biomarker, wherein, described micro-fluidic chip comprises glass-chip substrate, described glass-chip substrate is provided with microfluidic channel, is provided with soft magnetism microarray in microfluidic channel.
Preferably, described soft magnetism microarray is made up of Rhometal cylinder, and adjacent two row Rhometal cylinders are staggered.
More preferably, the distance of described adjacent two row Rhometal cylinders is 100 μ m, and the diameter of Rhometal cylinder is 30 μ m, and the distance in every row between adjacent two Rhometal cylinders is 50 μ m.
Preferably, described tumor stem cell biomarker comprises at least one of EpCAM, CD133, CD90, CD24, CD13, ICAM-1, SALL4, CD44 and ALDH.
The present invention also provides the first using method of the above-mentioned circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, it is characterized in that, concrete steps comprise:
Step 1: cracking peripheral red blood cells, centrifugal, remove supernatant, resuspended acquisition Peripheral Blood Nucleated Cells suspension;
Step 2: can mix Peripheral Blood Nucleated Cells suspension with the immunomagnetic beads that is marked with tumor stem cell biomarker of circulating tumor stem cell specific binding, and hatch, and obtain being marked with the Peripheral Blood Nucleated Cells suspension of immunomagnetic beads;
Step 3: apply external magnetic field in micro-fluidic chip bottom, the Peripheral Blood Nucleated Cells suspension that is marked with immunomagnetic beads is pumped in the microfluidic channel of micro-fluidic chip;
Step 4: by pumping in the microfluidic channel (2) of micro-fluidic chip for the fluorescence antibody of tumor stem cell biomarker accordingly, hatch microscopic examination circulating tumor stem cell;
Step 5: remove micro-fluidic external magnetic field, reclaim circulating tumor stem cell to carry out downstream molecules biological experiment.
The present invention also provides the another kind of using method of the above-mentioned circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, it is characterized in that, concrete steps comprise:
Steps A: cracking peripheral red blood cells, centrifugal, remove supernatant, resuspended acquisition Peripheral Blood Nucleated Cells suspension;
Step B: by mixing Peripheral Blood Nucleated Cells suspension for the fluorescence antibody of tumor stem cell biomarker, hatch, obtain containing the Peripheral Blood Nucleated Cells suspension for the fluorescence antibody of tumor stem cell biomarker;
Step C: can correspondingly be mixed with the Peripheral Blood Nucleated Cells suspension for the fluorescence antibody of tumor stem cell biomarker with the immunomagnetic beads that is marked with tumor stem cell biomarker of circulating tumor stem cell specific binding, hatch, obtain being marked with the Peripheral Blood Nucleated Cells suspension of immunomagnetic beads;
Step D: apply external magnetic field in micro-fluidic chip bottom, the Peripheral Blood Nucleated Cells suspension that is marked with immunomagnetic beads is pumped in the microfluidic channel of micro-fluidic chip to microscopic examination circulating tumor stem cell;
Step e: remove micro-fluidic external magnetic field, reclaim circulating tumor stem cell to carry out downstream molecules biological experiment.
A line above in Fig. 2 has shown the flow process of the first using method of the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, and remainder has shown the flow process of the second using method of the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip.
Compared with prior art, the invention has the beneficial effects as follows:
The immunomagnetic beads that utilization of the present invention is marked with circulating tumor stem cell markers mixes peripheral blood and circulating tumor stem cell specific binding, catch circulating tumor stem cell by soft magnetism microarray in micro-fluidic chip, realize many marks classification and Detection of the rare circulating tumor stem cell of peripheral blood, remove demagnetization after externally-applied magnetic field, the circulating tumor stem cell of absorption is come off so that collect.The present invention combines immunomagnetic beads this two large advantage to the combination of cell high-efficient rate and micro-fluidic chip high flux Dynamical capture, can realize a sample feeding and get final product the multiple different classes of rare circulating tumor stem cell of Acquisition Detection, there is higher sensitivity and specificity, easy to operate quick, catch cell and be easy to collect, without features such as the anti-finishing processes of primary antibodie two of chip internal microchannel complexity.
Brief description of the drawings
Fig. 1 is microfluidic chip structure schematic diagram;
Fig. 2 is that the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip is used process flow diagram;
Fig. 3 is the circulation liver-cancer stem cell testing result figure to EpCAM, CD133, CD90, the CD24 positive.Upper right, red fluorescence mark EpCAM
+circulation liver-cancer stem cell, blue-fluorescence labeled cell core; Upper left, red fluorescence mark CD133
+circulation liver-cancer stem cell, green fluorescence mark CD45
+leucocyte, blue-fluorescence labeled cell core; Bottom right, green fluorescence mark CD90
+circulation liver-cancer stem cell, red fluorescence mark CD45
+leucocyte, blue-fluorescence labeled cell core; Lower-left, red fluorescence mark CD24
+circulation liver-cancer stem cell, green fluorescence mark CD45
+leucocyte, blue-fluorescence labeled cell core.
Fig. 4 is for use business to sell CellSearch simultaneously
tMcirculating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip in system and this patent scheme detects comparing result to 33 routine peripheral blood of patients with primary hepatocellular carcinoma EpCAM+ circulating tumor stem cells.
Embodiment
Illustrate the present invention below in conjunction with embodiment.
1, the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, composed of the following components:
(1) micro-fluidic chip, as shown in Figure 1, for microfluidic chip structure schematic diagram, described micro-fluidic chip micro-fluidic chip comprises glass-chip substrate 1, it is 50mm that described glass-chip substrate 1 is provided with length a, width b is the microfluidic channel 2 of 17mm, is provided with soft magnetism microarray in microfluidic channel 2.Described soft magnetism microarray is made up of Rhometal cylinder 3, and adjacent two row Rhometal cylinders 3 are staggered.The distance c of described adjacent two row Rhometal cylinders 3 is 100 μ m, and the diameter d of Rhometal cylinder 3 is 30 μ m, and the distance e in every row between adjacent two Rhometal cylinders 3 is 50 μ m.Rear row Rhometal cylinder 3 horizontal median axis are 25 μ m to the shortest vertical range f at previous column Rhometal cylinder 3 edges.
(2) can with the immunomagnetic beads that is marked with tumor stem cell biomarker of circulating tumor stem cell specific binding, comprise: immunomagnetic beads (the Catalog no.130-061-101.Miltenyi Biotec of anti-EpCAM, Germany), immunomagnetic beads (the Catalog no.130-096-253 of the immunomagnetic beads of anti-CD133 (Catalog no.130-050-801.Milteny Biotec), anti-CD90, Milteny Biotec) and the immunomagnetic beads (Catalog no.130-095-951, Milteny Biotec) of anti-CD24
(3) for the fluorescence antibody of tumor stem cell biomarker, comprise: fluorescence antibody (the Catalog no.130-091-253 of anti-EpCAM-PE, Milteny Biotec), fluorescence antibody (the Catalog no.130-080-801 of anti-CD133-PE, Milteny Biotec), fluorescence antibody (the Catalog no.130-095-953 of anti-CD90-FITC, Milteny Biotec), fluorescence antibody (the Catalog no.130-095403 of anti-CD24-PE, Milteny Biotec), fluorescence antibody CD45-FITC (the Catalog no.555482 of leucocyte mark, BD bioscience) and CD45-PE (Catalog no.561866, BD bioscience),
2, prepare micro-fluidic chip:
(1) sputtered with Ti/Cu Seed Layer on glass substrate, then obtain the formpiston according to the soft magnetism microarray of software simulation design by positive glue photoetching, then carry out electroforming, obtain the soft magnetism microarray being made up of Rhometal cylinder 3 as shown in Figure 1, the material of Rhometal cylinder 3 was made up of (nickel and iron form according to the mass percent of 80: 20).
(2) remove Cu/Ti Seed Layer by the method for chemical corrosion, obtain transparent glass-chip; Then scribing, obtains single glass-chip substrate 1;
(3) by the photoetching of the suprabasil SU8 glue of silicon chip, obtain the former of needed microfluidic channel, then, by PDMS rapid shaping, prepare microfluidic channel 2;
(4) finally adopt conventional method by microfluidic channel 2 and glass-chip substrate 1 bonding that pressurizes, make micro-fluidic chip.
3, use the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, four kinds of unlike signal thing positives in Peripheral Blood of Patients with Hepatocellular Carcinoma (are comprised to EpCAM
+, CD133
+, CD90
+, CD24
+) circulation liver-cancer stem cell catch, concrete steps are:
Steps A: gather liver cancer patient 8ml peripheric venous blood, be divided into 4 parts, every part of 2ml, respectively with erythrocyte cracked liquid (the Red blood cell lysis solution10X of its 9X volume, Catalog no.130094183, Miltenyi Biotec) room temperature mixes 2 minutes, add equal-volume PBS damping fluid (PBS1X, pH=7.4, Catalog no.21-040-CVR, Corning, ) stop splitting red reaction, room temperature centrifugal 5 minutes with the centrifugal force of 350g, remove supernatant, 300 μ l PBS damping fluids (pH=7.4) are resuspended, obtain the whole karyocytes of peripheral blood and (comprise tumour cell, leucocyte, endothelial cell etc.).4 part of 300 μ l Peripheral Blood Nucleated Cells suspension of final acquisition.
Step B: by the fluorescence antibody of anti-EpCAM-PE (the same) with ratio 1: 100, the fluorescence antibody of leucocyte mark CD45-FITC (the same) with first part 300 μ l Peripheral Blood Nucleated Cells suspension mixes with ratio at 1: 200, by the fluorescence antibody of anti-CD133-PE with ratio 1: 100, the fluorescence antibody of leucocyte mark CD45-FITC (the same) with second part 300 μ l Peripheral Blood Nucleated Cells suspension mixes with ratio at 1: 200, by the fluorescence antibody of anti-CD90-FITC with ratio 1: 100, the fluorescence antibody of leucocyte mark CD45-PE (the same) with three part 300 μ l Peripheral Blood Nucleated Cells suspension mixes with ratio at 1: 200, by the fluorescence antibody of anti-CD24-PE with ratio 1: 100, the fluorescence antibody of leucocyte mark CD45-FITC (the same) with four part 300 μ l Peripheral Blood Nucleated Cells suspension mixes with ratio at 1: 200, incubated at room 1 hour, room temperature centrifugal 5 minutes with 350g centrifugal force, remove supernatant, add respectively 300 μ l PBS damping fluids (pH=7.4) resuspended, obtain 4 part of 300 μ l and contain the Peripheral Blood Nucleated Cells suspension for the fluorescence antibody of tumor stem cell biomarker,
Step C: get 4 kinds of anti-EpCAMs, CD133, CD90, 4 part of 300 μ l that the each 100 μ l of CD24 immunomagnetic beads and step B obtain contains for the Peripheral Blood Nucleated Cells suspension of the fluorescence antibody of tumor stem cell biomarker and fully mixes respectively (the tumor stem cell mark fluorescence antibody using in detecting with this in detection for the immunomagnetic beads of liver-cancer stem cell mark is corresponding), hatch 30 minutes for 4 DEG C, add 1mlPBS damping fluid (pH=7.4), 4 DEG C of the centrifugal force with 350g are centrifugal 5 minutes, remove supernatant, add 1ml PBS damping fluid (pH=7.4) resuspended, obtain being marked with the Peripheral Blood Nucleated Cells suspension of immunomagnetic beads,
Step D: get 4 micro-fluidic chips, apply respectively external magnetic field (Ru-Fe-Mn permanent magnet) in micro-fluidic chip bottom, the Peripheral Blood Nucleated Cells suspension that 4 parts of 1ml that step C obtains are marked with immunomagnetic beads pumps at the uniform velocity respectively in the microfluidic channel 2 of micro-fluidic chip with the speed of 2ml/hr.The EpCAM that fluorescence microscopy Microscopic observation is caught
+, CD133
+, CD90
+, CD24
+circulation liver-cancer stem cell.(as shown in Figure 3)
Step e: remove micro-fluidic external magnetic field, reclaim circulating tumor stem cell to carry out downstream molecules biological experiment.
Detect in the present embodiment the EpCAM of 33 peripheral blood of patients with primary hepatocellular carcinomas
+, CD133
+, CD90
+, CD24
+circulation liver-cancer stem cell.Also gathered another part of 7.5ml peripheral blood from these 33 patient's peripheral veins simultaneously, and the circulating tumor cell detection system (CellSearch that uses current business to sell
tM, Johnson & Johnson, USA) and these 33 parts of peripheral bloods are carried out to EpCAM
+circulating tumor stem cell detects.Experimental result shows: using the micro-fluidic chip detection EpCAM+ circulating tumor stem cell recall rate in the present invention program is 75.76%, and CellSearch
tMthe recall rate of system is 54.54%.(as shown in Figure 4).In addition CellSearch
tMsystem only can detect a kind of circulating tumor stem cell of mark.Therefore the present invention program's micro-fluidic chip circulating tumor stem cell detector efficiency is significantly higher than current business's sale system, and can realize multiple markers circulating tumor stem cell and detect.
1, the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, forms identical with embodiment 1:
2, micro-fluidic chip is prepared with embodiment 1.
3, the blood sample of Hep3B cell line is mixed in preparation, the blood sample that simulation contains circulating tumor stem cell:
(1) Hep3B cell line is (purchased from Chinese Academy of Sciences's Shanghai Life Science Park, hepatocellular carcinoma tumour cell, the Hep3B cell positive rate of expressing EpCAM, CD133, CD90 and CD24 is respectively 99.8% point, 98%, 0.2% and 1%, the circulating tumor cell cell using Hep3B cell as the method for mixing in the present embodiment).Get 4 × 10
7hep3B cell, by every part 1 × 10
7cell respectively with following fluorescence antibody with 1: 100 mixed C D24-PE (Catalog no.130-095-403, Milteny Biotec), CD90-FITC (Catalog no.130-095-953, Milteny Biotec), EpCAM-PE (Catalog no.130-091-253, Milteny Biotec), CD133-PE (Catalog no.130-080-801, Milteny Biotec), hatch 15min for 4 DEG C, use flow cytometer (BD FACSAria, BD Biosciences) sorting EpCAM respectively
+, CD133
+, CD90
+and CD24
+each 1000 of Hep3B cell, use respectively 1ml PBS (PBS1X, Catalog no.21-040-CVR, Corning) resuspended.Get 4 Eppendorf pipes, in each pipe, put into the each 100 μ l of above-mentioned four kinds of Hep3B cell suspensions (each pipe contains 100 Hep3B cells), separately get 4 Eppendorf pipes, in each pipe, put into the each 10 μ l of above-mentioned four kinds of Hep3B cell suspensions (each pipe contains 10 Hep3B cells).
Above-mentioned four kinds of Hep3B cell suspensions that prepare are mixed respectively to 1ml healthy human peripheral blood, obtain respectively 100 EpCAM
+hep3B/1ml peripheral blood sample, 100 CD133
+hep3B/1ml peripheral blood sample, 100 CD90
+hep3B/1ml peripheral blood sample and 100 CD24
+hep3B/1ml peripheral blood sample.10 EpCAM
+hep3B/1ml peripheral blood sample, 10 CD133
+hep3B/1ml peripheral blood sample, 10 CD90
+hep3B/1ml peripheral blood sample and 10 CD24
+hep3B/1ml peripheral blood sample
4, use the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, four kinds of unlike signal thing positives in blood sample (are comprised to EpCAM
+, CD133
+, CD90
+, CD24
+) circulation liver-cancer stem cell catch, concrete steps are:
Step (1): by the above-mentioned blood sample preparing, respectively with erythrocyte cracked liquid (the Red blood cell lysis solution10X of its 9X volume, Catalog no.130094183, Miltenyi Biotec) room temperature mixes 2 minutes, add equal-volume PBS damping fluid (PBS1X, pH=7.4Catalog no.21-040-CVR, Corning) stop splitting red reaction, room temperature centrifugal 5 minutes with the centrifugal force of 350g, remove supernatant, 300 μ lPBS damping fluids (pH=7.4) are resuspended, obtain the whole karyocytes of peripheral blood and (comprise tumour cell, leucocyte, endothelial cell etc.).8 part of 300 μ l Peripheral Blood Nucleated Cells suspension of final acquisition.
Step (2): get anti-EpCAM, CD133, CD90, 8 part of 300 μ l Peripheral Blood Nucleated Cells suspension that the each 100 μ l of CD24 immunomagnetic beads and step B obtain fully mix respectively (for the immunomagnetic beads of liver-cancer stem cell mark in detection, use separately and detect with this in the tumor stem cell mark fluorescence antibody that uses corresponding), hatch 30 minutes for 4 DEG C, add 1ml PBS damping fluid (pH7.4), 4 DEG C of the centrifugal force with 350g are centrifugal 5 minutes, remove supernatant, add 1ml PBS damping fluid (pH=7.4) resuspended, obtain being marked with the Peripheral Blood Nucleated Cells suspension of immunomagnetic beads.
Step (3): get 8 micro-fluidic chips, apply respectively external magnetic field (Ru-Fe-Mn permanent magnet) in micro-fluidic chip bottom, the Peripheral Blood Nucleated Cells suspension that 8 parts of 1ml is marked with to immunomagnetic beads pumps at the uniform velocity respectively in the microfluidic channel 2 of micro-fluidic chip with the speed of 2ml/hr.The EpCAM that fluorescence microscopy Microscopic observation is caught
+, CD133
+, CD90
+, CD24
+circulation liver-cancer stem cell.
Step (4): the fluorescence antibody of anti-EpCAM-PE is added to first part of 600 μ l PBS damping fluid (pH7.4) with the ratio of 1: 100 and the fluorescence antibody of leucocyte mark CD45-PE together with the ratio of 1: 200, the fluorescence antibody of anti-CD133-PE is added to second part of 600 μ l PBS damping fluid (pH7.4) with the ratio of 1: 100 and the fluorescence antibody of leucocyte mark CD45-PE together with the ratio of 1: 200, the fluorescence antibody of anti-CD90-FITC is added to the 3rd part of 600 μ l PBS damping fluid (pH7.4) with the ratio of 1: 100 and the fluorescence antibody of leucocyte mark CD45-PE together with the ratio of 1: 200, the fluorescence antibody of anti-CD24-PE is added to the 4th part of 600 μ l PBS damping fluid (pH=7.4) with the ratio of 1: 100 and the fluorescence antibody of leucocyte mark CD45-PE together with the ratio of 1: 200, under the environment of room temperature lucifuge, pump into respectively with the speed of 1ml/hr in the microfluidic channel 2 of the micro-fluidic chip that contains corresponding immunomagnetic beads, until 600 μ l all pump into (needing 36 minutes), whole operating process keeps bottom externally-applied magnetic field to exist, microscopic examination circulating tumor stem cell.
Step (5): remove micro-fluidic external magnetic field, reclaim circulating tumor stem cell to carry out downstream molecules biological experiment.
The EpCAM that this kit is caught
+, CD133
+, CD90
+and CD24
+hep3B cell quantity be respectively: mix 100/ml cell, the capture rate of every kind of mark cell is up to 99%, 98%, 98% and 99%, mix 10/ml cell, the capture rate of every kind of mark cell is up to 100%, 80%, 90% and 90%.The MACS cell enrichment system (QuadroMACS that uses current business to sell
tMseparator and Starting Kits, Miltenyi Biotec, Germany), according to product description, identical blood sample is processed, mix 100/ml cell, the capture rate of every kind of mark mark Hep3B cell is only respectively 32%, 25%, 30%, 36%.Mix 10/ml cell, four kinds of mark mark Hep3B cell organic efficiencies are zero.Experimental result shows, in the present invention program in classification capture rate, the especially peripheral blood of micro-fluidic chip human peripheral blood unlike signal thing circulating tumor cell denier cell (10/ml of <) capture rate apparently higher than current business's sale system.
Claims (6)
1. the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip, it is characterized in that, comprise micro-fluidic chip, at least one can with the immunomagnetic beads that is marked with tumor stem cell biomarker of circulating tumor stem cell specific binding and at least one fluorescence antibody for tumor stem cell biomarker, wherein, described micro-fluidic chip comprises glass-chip substrate (1), described glass-chip substrate (1) is provided with microfluidic channel (2), and microfluidic channel is provided with soft magnetism microarray in (2).
2. the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip as claimed in claim 1, it is characterized in that, described soft magnetism microarray is made up of Rhometal cylinder (3), and adjacent two row Rhometal cylinders (3) are staggered.
3. the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip as claimed in claim 2, it is characterized in that, the distance (a) of described adjacent two row Rhometal cylinders (3) is 100 μ m, the diameter (b) of Rhometal cylinder (3) is 30 μ m, and the distance (c) in every row between adjacent two Rhometal cylinders (3) is 50 μ m.
4. the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip as claimed in claim 1, it is characterized in that, described tumor stem cell biomarker comprises at least one of EpCAM, CD133, CD90, CD24, CD13, ICAM-1, SALL4, CD44 and ALDH.
5. the using method of the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip described in any one in claim 1-4, is characterized in that, concrete steps comprise:
Step 1: cracking peripheral red blood cells, centrifugal, remove supernatant, resuspended acquisition Peripheral Blood Nucleated Cells suspension;
Step 2: can mix Peripheral Blood Nucleated Cells suspension with the immunomagnetic beads that is marked with tumor stem cell biomarker of circulating tumor stem cell specific binding, and hatch, and obtain being marked with the Peripheral Blood Nucleated Cells suspension of immunomagnetic beads;
Step 3: apply external magnetic field in micro-fluidic chip bottom, the Peripheral Blood Nucleated Cells suspension that is marked with immunomagnetic beads is pumped in the microfluidic channel (2) of micro-fluidic chip;
Step 4: by pumping in the microfluidic channel (2) of micro-fluidic chip for the fluorescence antibody of tumor stem cell biomarker accordingly, hatch microscopic examination circulating tumor stem cell;
Step 5: remove micro-fluidic external magnetic field, reclaim circulating tumor stem cell to carry out downstream molecules biological experiment.
6. the using method of the circulating tumor stem cell detection kit based on magnetic bead and micro-fluidic chip described in any one in claim 1-4, is characterized in that, concrete steps comprise:
Steps A: cracking peripheral red blood cells, centrifugal, remove supernatant, resuspended acquisition Peripheral Blood Nucleated Cells suspension;
Step B: by mixing Peripheral Blood Nucleated Cells suspension for the fluorescence antibody of tumor stem cell biomarker, hatch, obtain containing the Peripheral Blood Nucleated Cells suspension for the fluorescence antibody of tumor stem cell biomarker;
Step C: can correspondingly be mixed with the Peripheral Blood Nucleated Cells suspension for the fluorescence antibody of tumor stem cell biomarker with the immunomagnetic beads that is marked with tumor stem cell biomarker of circulating tumor stem cell specific binding, hatch, obtain being marked with the Peripheral Blood Nucleated Cells suspension of immunomagnetic beads;
Step D: apply external magnetic field in micro-fluidic chip bottom, the Peripheral Blood Nucleated Cells suspension that is marked with immunomagnetic beads is pumped in the microfluidic channel (2) of micro-fluidic chip to microscopic examination circulating tumor stem cell;
Step e: remove micro-fluidic external magnetic field, reclaim circulating tumor stem cell to carry out downstream molecules biological experiment.
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Application publication date: 20140618 Assignee: Shanghai Dunhui Medical Technology Development Co., Ltd. Assignor: Zhongshan Hospital Affiliated to Fudan University Contract record no.: 2018310000061 Denomination of invention: Circulating tumor stem cell detection kit based on magnetic beads and microfluidic chip Granted publication date: 20170201 License type: Exclusive License Record date: 20181203 |