CN103087899A - Aptamer-based microfluidic chip capable of capturing cancer cells and preparation thereof as well as separation method of cancer cells - Google Patents
Aptamer-based microfluidic chip capable of capturing cancer cells and preparation thereof as well as separation method of cancer cells Download PDFInfo
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- CN103087899A CN103087899A CN2013100172624A CN201310017262A CN103087899A CN 103087899 A CN103087899 A CN 103087899A CN 2013100172624 A CN2013100172624 A CN 2013100172624A CN 201310017262 A CN201310017262 A CN 201310017262A CN 103087899 A CN103087899 A CN 103087899A
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Abstract
The invention discloses an aptamer-based microfluidic chip capable of capturing cancer cells, which is formed by reversibly sealing an upper-layer polymer chip and a lower-layer slide carrier, wherein a cell capturing channel is arranged between the upper-layer polymer chip and the lower-layer slide carrier; the bottom surface of the channel is rough; and avidin is fixed on the bottom surface. The preparation of the microfluidic chip comprises the steps of making a chip die by photoetching, and casting to obtain a polymer chip; processing the slide carrier with hydrofluoric acid and bonding with the polymer chip; and fixing avidin in the cell capturing channel to obtain a microfluidic chip. The microfluidic chip disclosed by the invention can be used for separating cancer cells; and the separation comprises the steps of adding biotin-modified aptamer into a sample, and incubating; and leading the mixed solution after the incubation into the microfluidic chip, wherein the target cancer cells in the mixed solution are combined with the avidin through the biotin to realize separation of the target cancer cells. The microfluidic chip disclosed by the invention has the advantages of high cell capturing efficiency, convenience in operation, strong generality, low cost and the like.
Description
Technical field
The present invention relates to a kind of cell separation technology, relate in particular to a kind of micro-fluidic chip and separation method that cancer cells separates that can be used for.
Background technology
Cancer also claims malignant tumour, and it is because controlling the not normal disease that causes of growth and proliferation of cell mechanism.Cancer cells except grow out of control, arround also can local invading, healthy tissues is even transferred to other parts of health, serious threat human health via body-internal-circulation system or lymphsystem.Catching of cancer cells especially splits away off from primary cancer cell tissue the catching of circulating cancer cells that enters human peripheral blood and tissue, for the early diagnosis of cancer, the assessment of result for the treatment of, extremely important meaning arranged.In the past few decades, people have developed a variety of in order to catching the technology of cancer cells, as: fluorescent immune method, magnetic immuno method etc.Yet, the instrument costliness that these technology are required, working method is complicated, promotes difficulty large.Therefore, to catch instrument be very significant to a kind of cancer cells small-sized, cheap, simple to operate of exploitation.
The micro-fluidic chip technology is low due to cost of manufacture, reagent consumption less, detection speed is fast, the functional integration advantages of higher more and more is subject to the researchist concern.People have progressively utilized micro-fluidic chip to realize technology such as high flux screening, counting and sign to circulating cancer cells.Faley S etc. utilizes the polydimethylsiloxane (Polydimethylsiloxane of 440 18 μ m * 18 μ m * 10 μ m, PDMS) the microstructure capture array has successfully realized (the Faley S that catches to the CD4+T cell, Seale K, Hughey J et al, Microfluidic Platform for Real-Time Signaling Analysis of Multiple Single T Cells in Parallel, Lab on a Chip, 2008,8,1700-1712).The wide small slit of this design side direction 2 μ m makes PDMS easily rupture when the demoulding, and chip is not high to the selectivity of cell capture in addition, the red corpuscle in whole blood, white corpuscle and cancer cells can be caught simultaneously.These methods basically all are based on the physical properties such as cell size or separate based on the identification of antigen antibody interaction, so all could not realize existing high-level efficiency, again catching of high specific arranged.
Aptamer (aptamer) be a kind of screening obtains from the DNA/RNA library of synthetic can high-affinity and the single stranded oligonucleotide of being combined with target molecules with high specificity.Nineteen ninety Gold study group use the in-vitro screening technology obtain can with the RNA of T4 archaeal dna polymerase specific binding, and this technology is defined as the aglucon phyletic evolution technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX) of index concentration.The aptamer that screening obtains based on SELEX has than the better chemical property of antibody, can be efficiently, specifically in conjunction with the plurality of target material, have the advantages such as the preparation modification is easy fast, good stability, nontoxicity, non-immunogenicity, demonstrating huge application prospect aspect biomedical fundamental research and clinical diagnosis and treatment.the use aptamers such as Joseph A. Phillips have been realized separation and enrichment (the Joseph A. Phillips of cancer cells effectively as capture probe, Ye Xu, Zheng Xia et al Enrichment of cancer cells using aptamers immobilized on a microfluidic channel Anal. Chem. 2009, 81, 1033-1039), but the method is because chip structure is simple, can not solve well the impact of laminar flow, in addition because aptamer is fixed on chip in advance, itself and cells contacting probability are little, this makes the capture rate under lower concentration not high.
Therefore, in the urgent need to developing a kind of simple, easy to operate cell capture sorting chip of making, to realizing efficiently and with high specificity catching the target of micro-cancer cells in sample.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, the micro-fluidic chip that provide that a kind of cell capture efficient is high, manufacture craft is simple, easy and simple to handle, highly versatile, cost are low, can catch cancer cells in whole blood also provides a kind of separation method of the cancer cells fast and accurately based on this micro-fluidic chip.
for solving the problems of the technologies described above, the technical scheme that the present invention proposes is a kind of micro-fluidic chip of the caught cancer cells based on aptamer, described micro-fluidic chip is by upper strata polydimethylsiloxane (Polydimethylsiloxane, PDMS) the reversible sealing-in of slide carrier of polymer chip and lower floor forms, described micro-fluidic chip is provided with injection port and outlet, be provided with the cell capture passage of snakelike shape between described PDMS polymer chip and slide carrier, described cell capture passage is communicated with described injection port and outlet, the interior upper surface of described cell capture passage is provided with outstanding from top to down herringbone structure, the basal surface of described cell capture passage is rough slide carrier surface, be fixed with avidin on the basal surface of described cell capture passage.
In above-mentioned micro-fluidic chip, described rough slide carrier surface is to obtain through hydrofluoric acid corrosion treatment aftershaping.
As a total technical conceive, the present invention also provides a kind of preparation method of micro-fluidic chip, comprises the following steps:
(1) PDMS polymer chip: first draw PDMS polymer chip reticle pattern with mapping software, and use the high-resolution laser printer to be printed on to make mask blank on transparent film, then utilize this mask blank and make by photoetching process to obtain the chip mould;
(2) PDMS casting: with aggressiveness before PDMS and solidifying agent routinely mass ratio (for example 10: 1) carry out physical mixed, fully stir, cast in after vacuum outgas on the chip mould that above-mentioned steps (1) makes, cooling after baking and curing, obtain the PDMS polymer chip;
(3) hydrofluoric acid treatment: hydrofluoric acid solution evenly is coated in the glass surface of a cleaning, standing for some time (washes after 5s～5min), dries up with nitrogen, obtain the slide carrier; By adopting the hydrofluoric acid etching glass surperficial, can increase the roughness of glass surface, improve the bottom surface area of cell capture passage, and then improve the modification density of avidin, further destroy the contact probability that laminar flow increases cell and substrate, reach the purpose that increases the target cell capture rate;
(4) bonding: the slide carrier pressurization reversible keying that the PDMS polymer chip that step (2) is made and step (3) make obtains work in-process;
(5) modification of avidin: the phosphoric acid buffer that will contain avidin is passed in half-finished cell capture passage by half-finished injection port, and it is hatched in the cell capture passage, then rinses with binding buffer liquid, makes micro-fluidic chip.
Above-mentioned preparation method, in described step (2), the temperature of baking and curing is preferably 70 ℃～90 ℃, and the hot baking time is preferably 2～6 hours.
Above-mentioned preparation method, in described step (3), the volume fraction of hydrofluoric acid solution is preferably 10%～40%.
As a total technical conceive, the present invention also provides a kind of separation method of cancer cells, comprises the following steps:
(1) add aptamer solution in the sample that contains targeted cancerous cells, mixing solutions is placed on ice hatches; The aptamer that contains in described aptamer solution is to carry out the aptamer that specific reaction is combined with targeted cancerous cells, and this aptamer generally is selected from one or more in DNA, RNA, and terminal modified vitamin H is arranged in 3 ' of this aptamer sequence; Aptamer based on SELEX screening obtains has than traditional better chemical property of capture probe antibody, can be efficiently, specifically in conjunction with the plurality of target material, have preparation modify easy fast, the advantages such as good stability, nontoxicity, non-immunogenicity;
(2) mixing solutions after hatching in step (1) is passed in the injection port of above-mentioned micro-fluidic chip, mixing solutions is flowed in described cell capture passage, hatch under room temperature, the targeted cancerous cells that contains in mixing solutions is by the association reaction of described vitamin H and avidin, be captured to the slide carrier surface of described micro-fluidic chip, other compositions in mixing solutions flow out by described outlet, realize the separation to targeted cancerous cells.
In above-mentioned separation method, described targeted cancerous cells comprises various types of cancer cells, preferably refers to lymphocytic cancer cell, lung carcinoma cell or liver cancer cell; Described sample even can be the human whole blood sample of complexity.By adopting separation method of the present invention that the sample that contains targeted cancerous cells is separated, this not only provides means and prerequisite for the follow-up scientific research of various cancer cells, and can effectively realize the quantitative and qualitative analysis of cancer cells is detected by prior aries such as fluorometric analyses.
The separation method of above-mentioned cancer cells, in described step (2), mixing solutions preferably is passed in the injection port of micro-fluidic chip with the flow velocity of 0.5 mL/h～3 mL/h; The time of hatching under room temperature is preferably 5min～15min.
Compared with prior art, the invention has the advantages that: micro-fluidic chip of the present invention not only preparation method is quick, the avidin fixing means is simple, and by the micro-fluidic chip technology is combined with the aptamer detection technique, can use aptamer targetedly according to different cancer cells to be checked, detection sensitivity is higher, and specificity is better.The micro-fluidic chip that the present invention makes can be used for carrying out catching and separating of cell, especially cancer cells from the sample that contains the complex cell composition (for example whole blood or tissue), has characteristics easy and simple to handle, that integrated level is high.Because mould, the chip itself of micro-fluidic chip are all reusable, and need not complicated finishing process, so the preparation cost of product of the present invention is lower, has better economy and application and popularization value.
Description of drawings
Fig. 1 is the structural representation of micro-fluidic chip in the embodiment of the present invention.
Fig. 2 is that the targeted cancerous cells fluorescence of cancer cells separation method of the present invention in embodiment is caught result.
Fig. 3 is that the targeted cancerous cells fluorescence of control sample cancer cells separation method in embodiment is caught result.
Fig. 4 is that the glass surface of micro-fluidic chip in the embodiment of the present invention is corroded the stereoscan photograph of front surface pattern by hydrofluoric acid.
Fig. 5 is that the glass surface of micro-fluidic chip in the embodiment of the present invention is corroded the stereoscan photograph of rear surface pattern by hydrofluoric acid.
Fig. 6 is that the target cell fluorescence that in the embodiment of the present invention, glass surface is carried out obtaining after the hydrofluoric acid corrosion treatment is caught figure as a result.
Fig. 7 does not carry out to glass surface the target cell fluorescence that the hydrofluoric acid corrosion treatment obtains in the embodiment of the present invention to catch figure as a result.
Marginal data:
1, injection port; 2, outlet; 3, cell capture passage; 4, PDMS polymer chip; 5, slide carrier; 6, avidin; 7, basal surface.
Embodiment
The invention will be further described with concrete preferred embodiment below in conjunction with Figure of description, but protection domain not thereby limiting the invention.
Embodiment:
a kind of micro-fluidic chip of the caught cancer cells based on aptamer of the present invention as shown in Figure 1, this micro-fluidic chip is formed by the ventilative PDMS polymer chip 4 of upper strata light-permeable and the reversible sealing-in of slide carrier 5 of lower floor, micro-fluidic chip is provided with injection port 1 and outlet 2, be provided with the cell capture passage 3 of snakelike shape between PDMS polymer chip 4 and slide carrier 5, cell capture passage 3 is communicated with injection port 1 and outlet 2, the interior upper surface of cell capture passage 3 is provided with outstanding from top to down herringbone structure, the basal surface 7 of cell capture passage 3 is rough slide carrier surface (referring to the partial enlarged drawing in Fig. 1 and Fig. 4), be fixed with avidin 6 on the basal surface of cell capture passage 3.
In above-mentioned micro-fluidic chip, rough slide carrier surface is to obtain through hydrofluoric acid corrosion treatment aftershaping.
The preparation method of the micro-fluidic chip of the present embodiment comprises the following steps:
(1) PDMS polymer chip: at first make PDMS polymer core die, use AutoCAD Software on Drawing PDMS polymer chip reticle pattern, and use high-resolution laser printer (resolving power 12000 dpi) to be printed on to make mask blank on transparent film, then utilize this mask blank (in Dalian Inst of Chemicophysics, Chinese Academy of Sciences) and make SU-8 negative photoresist chip mould by photoetching process;
(2) PDMS casting: with aggressiveness before PDMS and the solidifying agent (trade mark: sygard 184, the product of Dow corning company) carry out physical mixed by 10 ︰ 1 mass ratioes, through ultrasonic, abundant stirring, cast in after the steps such as vacuum outgas 20min on the sun version chip mould that above-mentioned steps (1) makes, 80 ℃ of heat were dried by the fire 2 hours, naturally cooling obtains PDMS polymer chip 4;
(3) hydrofluoric acid treatment: be the glass surface that 10% hydrofluoric acid solution evenly is coated in a cleaning with volume fraction, naturally process the 60s afterwash, dry up with nitrogen, obtain slide carrier 5; The forward and backward glass surface of hydrofluoric acid treatment is distinguished as shown in Figure 4 and Figure 5; By Fig. 4 and Fig. 5 as seen, the glass surface opposed flattened before processing, the glass surface after processing shows significantly uneven; Adopt the hydrofluoric acid etching glass surperficial, can increase the roughness of glass surface, improve the surface-area of channel bottom, improve the modification density of avidin, further increase the contact probability that laminar flow increases cell and substrate, reach the purpose that increases the target cell capture rate;
(4) bonding: the PDMS polymer chip 4 that under dustfree environment, step (2) is made is carefully taken off from the chip mould, and the slide carrier 5 that itself and step (3) make is carried out respectively the spend the night sterilising treatment of irradiation of ultraviolet, then the reversible keying that pressurizes obtains work in-process;
(5) modification of avidin: the phosphoric acid buffer that will contain the 1mg/mL avidin passes in above-mentioned half-finished cell capture passage by injection port, make it be full of the cell capture passage, and stop 30s, then (contain DPBS with the flushing of binding buffer liquid, 4.5 g/L glucose, 5 mmol/L MgCl
2, 0.1 mg/mL yeast tRNA, 1 mg/mL BSA, 10% FBS), make the micro-fluidic chip finished product.
A kind of separation method of cancer cells of the present invention, the micro-fluidic chip that it uses above-mentioned the present embodiment specifically comprises the following steps:
(1) get appropriate people's whole blood sample, add a certain amount of targeted cancerous cells through the fluorescent dye mark (selecting CCRF-CEM people's acute lymphoblastic leukemia cell in the present embodiment), add aptamer solution (making its concentration in mixing sample solution is 1 μ mol/L) in the people's whole blood sample that contains targeted cancerous cells, mixing solutions is placed in hatches 10min on ice; The aptamer that contains in aptamer solution in the present embodiment is can carry out the aptamer Sgc8(that specific reaction is combined with targeted cancerous cells to obtain by the SELEX technology screening, nucleotide sequence is as follows), and terminal modified vitamin H arranged in 3 ' of this aptamer sequence; This aptamer sequence is:
5’-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGATTTTTTTTTT-3’-biotin；
The aptamer that screening obtains based on SELEX, have than the better chemical property of traditional capture probe (as antibody), can be efficiently, specifically in conjunction with the plurality of target material, have preparation modify easy fast, the advantages such as good stability, nontoxicity, non-immunogenicity;
(2) mixing solutions after hatching in step (1) is passed in the injection port 1 of above-mentioned micro-fluidic chip, make mixing solutions flow rate with 2 mL/h in its cell capture passage 3, at room temperature hatch 7min, the targeted cancerous cells that contains in mixing solutions is by the association reaction of vitamin H and avidin, be captured on the basal surface 7 of slide carrier 5 of micro-fluidic chip, other compositions in mixing solutions flow out by outlet 2, realize the separation to targeted cancerous cells; 3 times of dcq buffer liquid to sample volume (contain DPBS, 4.5 g/L glucose, 5 mmol/L MgCl
2) rinse afterwards and detect and count under fluorescent microscope.
Consider that traditional method is first at first to pass into aptamer solution in micro-fluidic chip, be fixed in the bottom of micro-fluidic chip cell capture passage, and the present invention first evenly mixes aptamer solution with sample, make a large amount of aptamers be combined in the surface of targeted cancerous cells, after passing into micro-fluidic chip again, the area that cancer cells is combined with the avidin of chip base and probability can obviously improve, thereby improve capture rate.Based on this, choose one group of same sample as the contrast of above-mentioned the present embodiment cancer cells separation method, the separation method of its sepn process and the present embodiment cancer cells is similar, but in step (1), not first aptamer solution and sample mix to be hatched, but employing first is fixed on aptamer the basal surface of the cell capture passage 3 of micro-fluidic chip, more directly sample is passed in the cell capture passage 3 of micro-fluidic chip.The targeted cancerous cells fluorescence of the separation method that the present embodiment is above-mentioned and control sample is caught result respectively as shown in Figures 2 and 3.Comparison diagram 2 and Fig. 3 as seen, same testing sample, the isolated cancer cells quantity of the inventive method is obviously more than control sample, namely the separation method of the present invention method of comparing control sample has better sensitivity, the capture ability of cancer cells is stronger.
Based on this, choose in addition A, two groups of same sample of B, the above-mentioned the present embodiment cancer cells separation method of same employing separates, but the slide carrier of the micro-fluidic chip that uses in the B group does not pass through the hydrofluoric acid corrosion treatment, and the chip glass surface treatment that the present embodiment is above-mentioned and the targeted cancerous cells fluorescence of control sample are caught result respectively as shown in Figure 6 and Figure 7.Comparison diagram 6 and Fig. 7 as seen, same testing sample, after the present invention processed the slide carrier, the cancer cells capture ability significantly strengthened, detection sensitivity improves, and specificity is good, mould is reusable, need not complicated finishing process.
＜110〉Hunan University
＜120〉a kind of micro-fluidic chip of the caught cancer cells based on aptamer and the separation method of preparation and cancer cells thereof
<160> 1
<210> 1
<211> 51bp
<212> DNA
＜213〉artificial sequence
<400> 1
atctaactgc tgcgccgccg ggaaaatact gtacggttag attttttttt t 51
Claims (8)
1. micro-fluidic chip based on the caught cancer cells of aptamer, described micro-fluidic chip is formed by the reversible sealing-in of slide carrier of upper strata PDMS polymer chip and lower floor, described micro-fluidic chip is provided with injection port and outlet, be provided with the cell capture passage of snakelike shape between described PDMS polymer chip and slide carrier, described cell capture passage is communicated with described injection port and outlet, the interior upper surface of described cell capture passage is provided with outstanding from top to down herringbone structure, it is characterized in that: the basal surface of described cell capture passage is rough slide carrier surface, be fixed with avidin on the basal surface of described cell capture passage.
2. micro-fluidic chip according to claim 1, it is characterized in that: described rough slide carrier surface is to obtain through hydrofluoric acid corrosion treatment aftershaping.
3. the preparation method of a micro-fluidic chip comprises the following steps:
(1) PDMS polymer chip: first draw PDMS polymer chip reticle pattern with mapping software, and use the high-resolution laser printer to be printed on to make mask blank on transparent film, then utilize this mask blank and make by photoetching process to obtain the chip mould;
(2) PDMS casting: with aggressiveness before PDMS and solidifying agent routinely mass ratio carry out physical mixed, fully stirring casts in after vacuum outgas on the chip mould that above-mentioned steps (1) makes, and is cooling after baking and curing, obtains the PDMS polymer chip;
(3) hydrofluoric acid treatment: hydrofluoric acid solution evenly is coated in the glass surface of a cleaning, washes after standing, dry up with nitrogen, obtain the slide carrier;
(4) bonding: the slide carrier pressurization reversible keying that the PDMS polymer chip that step (2) is made and step (3) make obtains work in-process;
(5) modification of avidin: the phosphoric acid buffer that will contain avidin is passed in half-finished cell capture passage by half-finished injection port, and it is hatched in the cell capture passage, then rinses with binding buffer liquid, makes micro-fluidic chip.
4. preparation method according to claim 3, it is characterized in that: in described step (2), the temperature of baking and curing is 70 ℃～90 ℃, and the hot baking time is 2～6 hours.
5. according to claim 3 or 4 described preparation methods, it is characterized in that: in described step (3), the volume fraction of hydrofluoric acid solution is 10%～40%.
6. the separation method of a cancer cells comprises the following steps:
(1) add aptamer solution in the sample that contains targeted cancerous cells, mixing solutions is placed on ice hatches; The aptamer that contains in described aptamer solution is to carry out the aptamer that specific reaction is combined with targeted cancerous cells, and terminal modified vitamin H is arranged in 3 ' of this aptamer sequence;
(2) mixing solutions after hatching in step (1) is passed in the injection port of claim 1 or 2 described micro-fluidic chip, mixing solutions is flowed in described cell capture passage, at room temperature hatch, the targeted cancerous cells that contains in mixing solutions is by the combination of described vitamin H and avidin, be captured to the slide carrier surface of described micro-fluidic chip, other compositions in mixing solutions flow out by described outlet, realize the separation to targeted cancerous cells.
7. the separation method of cancer cells according to claim 6, it is characterized in that: described targeted cancerous cells comprises lymphocytic cancer cell, lung carcinoma cell or liver cancer cell, described sample is the human body whole blood sample.
8. the separation method of according to claim 6 or 7 described cancer cells is characterized in that: in described step (2), mixing solutions is that the flow velocity with 0.5 mL/h～3 mL/h is passed in the injection port of micro-fluidic chip; The time of hatching under room temperature is 5min～15min.
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