CN101250483A - Combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip - Google Patents
Combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip Download PDFInfo
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Abstract
The invention discloses a combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip and relates to a micro-fluidic chip which is applied in cell sample separation and enrichment, a dielectrophoresis (DEP ) splint combined type electrode is obtained through depositing gold film on the surface of glass, and the electrode can be connected with a corresponding external connected low pressure high frequency ac circuit to realize DEP separation and enrichment process control. The combined splint microelectrode which is provided by the invention designs that the functions of on-line separation and on-line enrichment of sample such as cells and the like are effectively integrated on a chip, which realizes organic combination between the micro-fluidic chip technique and the microelectrode principles, and the chip can be applied and can realize the separation and the enrichment of various cells and bacterium.
Description
Technical field:
The invention belongs to integrated little biochemical analysis systems technology, relate in particular to a kind of micro-fluidic chip that is used for cell separation and enrichment.
Background technology:
In fields such as chemistry and biology, based on the separation of the biological sample system of cell is a kind of technology of obtaining the target cell of character homogeneous from a large amount of non-homogeneous cell sample colony, be usually used in fields such as cytobiology, clinical medicine and medicament research and development, especially significant to detection, diagnosis and the treatment of disease.In the cell separation technology and method of micro-fluidic chip as technology platform, integrated micro-fluidic dielectrophoresis (DEP) chip has become the novel biochemical analytical technology that domestic and international researchist extremely pays close attention to.At present, correlative study is as the purpose that reaches cell enrichment by dielectrophoresis (DEP) means, and the dielectrophoresis separating chips is combined with microflow control technique, at home and abroad be still as the chip research of continuous separation, enrichment and the detection means of cell and be in the initial stage, remain to be goed deep into systematic research and exploitation.Nearly 2 years, most characteristic research mainly was the research at integration dielectric electrophoresis chip system and DEP biochemistry detection application facet.Tradition DEP chip research mainly concentrates on static separation enrichment and the microscopic observation aspect that realizes particulate by the design of array microelectrode, for fusion with microflow control technique and particle enrichment sepn process, the test of the qualitative, quantitative of particulate characteristic parameter in the DEP process, and integrated sensing detection device etc. still remains further investigation on chip; For the DEP chip application is realized detection by quantitative still in the primary stage in bacterium and cell system, people extremely pay close attention to the research at the more extensive target of biochemistry detection.
Summary of the invention:
Based on the deficiencies in the prior art, the present invention proposes a kind of combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip, design is also produced combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip and correspondent control circuits, and realized the enrichment of cell dielectric electrophoretic separation based on this chip, this chip can be applicable to separation rapidly, continuously and the enrichment of different sorts cell or bacterium.
Technical scheme of the present invention is as follows:
1. combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip, this chip includes substrate and is bonded in on-chip cover plate, on described substrate, adopt micro electronmechanical processing technology MEMS processes that two groups of clamping plate type electrodes are arranged, on this sheet, be processed with sample channel, separating pipe, enrichment pipeline, sample pool, buffered soln pond, sample inlet pool and waste liquid pool.
Described chip comprises two groups of clamping plate type electrodes, sample channel, separating pipe, the enrichment pipeline, sample pool, the buffered soln pond, sample inlet pool and waste liquid pool, described sample channel is " Y " type pipeline, its sample introduction end has three ports, one of them port links to each other with sample inlet pool, two other port links to each other with two buffered soln ponds respectively, the other end of sample channel is connected with described separating pipe head end, the tail end of separating pipe connects three small transfer lines, small transfer line is the example enrichment pipeline in the middle of described, example enrichment rear end of pipeline mouth connects sample pool, and other two small transfer lines of the tail end of described separating pipe are positioned at the both sides of example enrichment pipeline and link to each other with waste liquid pool; Described two groups of clamping plate type electrodes, wherein one group of arrangement of electrodes is in the bottom of separating pipe, be used to realize the continuous separation of cell mixing sample, another group arrangement of electrodes is in the bottom of enrichment pipeline, be used to realize the continuous enrichment of single cell sample, sample in the sample inlet pool enters into separating pipe with buffered soln in the buffered soln pond after sample channel mixes, separation and the enrichment that dielectrophoresis separates the realization sample takes place according to the dielectric characteristics of self in sample component under the effect of the inhomogeneous field that two groups of clamping plate type electrodes produce, realize that isolating different sample flows into sample pool and waste liquid pool respectively.
The present invention has the following advantages:
1. with the combining of dielectrophoresis principle and the success of micro-fluidic chip technology, set up the method for continuous dielectrophoresis cell separation and enrichment;
2. the combined splint microelectrode type micro-fluidic DEP chip that adopts the MEMS processes to make has that separation and concentration voltage is low, power consumption is few, does not damage advantage such as sample biological activity.
Description of drawings:
Fig. 1 is the micro-fluidic dielectrophoresis cell separation and enrichment chip synoptic diagram of clamping plate type of the present invention;
Fig. 2 is a chip piping drawing of the present invention;
Fig. 3 is the structural representation of first group of electrode;
Fig. 4 is the structural representation of second group of electrode;
Fig. 5 is a chip controls electrical block diagram of the present invention
Embodiment:
According to Figure of description, the technical solution of the present invention is further elaborated by specific embodiment below.
Fig. 1 is the micro-fluidic dielectrophoresis cell separation and enrichment chip synoptic diagram of clamping plate type of the present invention.Combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip structure comprises: first and second group clamping plate type electrode 4-1 and 4-2, sample introduction separating pipe 3 and enrichment pipeline 6.Referring to Fig. 2, sample introduction separating pipe 3 is the Y-piece road, and port is connected to feed liquor pond 2 and two buffered soln ponds 1, the separating pipe tail end has three small transfer lines, middle one is example enrichment pipeline 6, and port is connected to sample pool 7, and all the other two pipelines directly are connected to waste liquid pool 8.5 is electrode cables among the figure.
The combined splint microelectrode is divided into two groups, first group of clamping plate type electrode 41 is arranged in the bottom of sample introduction separating pipe 3, purpose is to realize the continuous separation of cell mixing sample, in conjunction with Fig. 3, specifically form the narrow electrode A of a wide electrode B that mediates and its both sides by three plane electrodes that are parallel to pipeline axial
1And A
2, and the spacing between the electrode equates; Second group of clamping plate type electrode 4-2 is arranged in the bottom of enrichment pipeline 6, and purpose is to realize the continuous enrichment of simple sample, and referring to Fig. 4, it is made up of 90 ° of two one-tenth and mutual disjunct plane clamping plate electrode.
Structural parameter at the separated region chip are: the length L of electrode A 1 and A2
1Be 500～2000 μ m, width L
2Be 5～20 μ m, the length of electrode B is L
3500～2000 μ m, width L
4Be 30～55 μ m, the spacing J between the electrode is 1～20 μ m, the separating pipe length L
XBe 800～2200 μ m, separating pipe width L
YBe 42～160 μ m (as shown in Figure 2).
Structural parameter at the rich region chip are: clamping plate electrode width L
5Be 5～20 μ m, electrode and duct wall angle at 45, electrode length L
6Be 20～28 μ m, the H of the minimum place of clamping plate interelectrode distance
1Be 5～10 μ m, maximum H
2Be 45～55 μ m, enrichment duct width L
X2Be 45～55 μ m, length L
X1Be not shorter than 300 μ m, chip microchannel overall depth is 10～50 μ m.
Chip microelectrode and microchannel adopt based on the processing technology of micro electronmechanical processing technology MEMS technology and make.Substrate adopts special-purpose optical glass material or the quartz that planeness is good, the surface has good light transmission and smooth finish.By at the golden film of substrate surface deposit thickness less than 1 μ m, obtain two groups of DEP clamping plate type electrodes, the cover plate of chip adopts polydimethylsiloxane (PDMS) sheet that microchannel is arranged, and both Direct Bonding obtain can be used for the DEP chip of cell separation and enrichment.The clamping plate type microelectrode adopts gold electrode, and at first titanizing film on sheet glass then, is transferred to the electrode structure figure that designs on the chip by photoetching, and degold takes off titanium then, finishes the decline making of electrode group of glass substrate train wheel bridge again behind hot setting.The making of microchannel on the cover plate, at first adopt polymkeric substance MEMS technology, utilize the SU-8 material to form the microchannel formpiston that requires, adopt the direct casting reverse mould of PDMS polymkeric substance then, after solidifying, peel off down the PDMS polymkeric substance cover plate that has the microchannel shape from formpiston, the degree of depth that forms as Fig. 1 is 30 μ m microchannel networks.At last,, directly will contain microelectrode glass substrate and the PDMS cover plate Direct Bonding that contains microchannel, form the micro-fluidic dielectrophoresis cell separation and enrichment chip of clamping plate built-up type by cleaning and drying up.
Fig. 5 is the pilot circuit of combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip, and the electrode on the chip substrate has all designed by gold wire, directly draws the connection hole of receiving pcb board.At separated region, electrode A 1 and A2 are connected to homopolarity, and itself and electrode B and external source are formed a pilot circuit; At rich region, the clamping plate electrode be heteropole and with and external source be connected to form another the group pilot circuit.Two groups of pilot circuits are connected by a two-way switch, and when switch was connected with the pilot circuit of separated region, the electrode of rich region kept trip condition; When switch was connected with the pilot circuit of rich region, the electrode of separated region kept trip condition.This can guarantee to realize that the designated area of the DEP chip that when given ordered pair is designed applies the control process of high frequency low voltage.
According to the dielectrophoresis separation principle, in the inhomogeneous field of space, be suspended in particulate in certain medium with respect to the induced dipole of buffered soln apart from different and produce electromigration.If the sample particulate is than the easy polarization of buffered soln, then it forms the forward dielectrophoresis to the zone migration of high electric field; On the contrary, if its polarization trend is weaker than the polarization of buffered soln, then it moves to low electric field region, forms the negative sense dielectrophoresis.Because there is different polarizatioies in different sample particulates, thereby can realize under the condition separating and enrichment identical adding.For this reason, the present invention is divided into separated region and rich region with the micro-fluidic dielectrophoresis cell separation and enrichment chip of clamping plate built-up type by function.
Three plane electrode As parallel with pipeline axial are arranged in separating pipe bottom at separated region
1, B and A
2, and the design correspondent peripheral circuit controls this three strip electrode, counter electrode applies voltage range at 0～20V, range of frequency is at the low pressure high frequency ac signal of 100KHz～20MHz, thus produce one with the vertical inhomogeneous field of pipeline axial.At the separation initial stage, sample enters into separating pipe with buffered soln after the Y-piece road mixes, and then dielectrophoresis takes place according to self dielectric characteristics and separates in sample component in inhomogeneous field, and in process of flowing, move towards the edge of electrode and the medullary ray of target B respectively, drive with fluidic, the sample of moving to target B position of center line enters into the enrichment pipeline of rich region, the sample of moving to electrode edge then flows to the small transfer line on rich region both sides, finally flows to waste liquid pool.
Enrichment duct bottom at rich region is furnished with two clamping plate electrodes that become 90 °, and design has correspondent peripheral circuit to control this two clamping plate electrodes, the clamping plate electrode is applied a voltage range at 0～20V, range of frequency produces an inhomogeneous field parallel with pipeline axial at the low pressure high frequency ac signal of 100KHz～20MHz.At the enrichment initial stage, the sample that enters rich region after separated region is realized separating will be subjected to the resistance of dielectrophoresis force herein, and then make the dispersive sample component to the further enrichment of medullary ray, and be arranged in rows by the center of clamping plate electrode, realize the sample enrichment process.
Below by specific embodiment, introduce this working of an invention process.At first carry out sample pre-treatments.Gather fresh blood and place the beaker that is added with antithrombotics, on slow speed of revolution whizzer with 2000r/min centrifugal 10 minutes then, abandoning supernatant added medical sodium-chlor isotonic solution (2.25g/250ml) then, sets up the method triplicate of stating and gets final product.Carry out the test of chip dielectrophoresis then.Before the sample feeding, adopt the pressure input mode that PBS buffered soln is passed into the pipeline from the buffering solution pool, adopt micro-injection pump that 20 μ l samples are injected sample pool then, two-way switch is communicated with the separated region pilot circuit, and apply voltage range at 1-10V, range of frequency is at the AC signal of 1-5MHz, sample is finished the dielectrophoresis motion in the separated region microchannel after, two-way switch is communicated with the rich region pilot circuit, and applying the low pressure high frequency ac signal of same range as, sample produces enrichment in oblique clamping plate zone.
Claims (7)
1. combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip, this chip includes substrate and is bonded in on-chip cover plate, it is characterized in that: on described substrate, adopt micro electronmechanical processing technology MEMS processes that two groups of clamping plate type electrodes are arranged, on this sheet, be processed with sample channel, separating pipe, enrichment pipeline, sample pool, buffered soln pond, sample inlet pool and waste liquid pool;
Described sample channel is " Y " type pipeline, its sample introduction end has three ports, in described three ports one links to each other with sample inlet pool, two other links to each other with the buffered soln pond, the rear end of sample channel is connected with described separating pipe head end, and the tail end of separating pipe connects three small transfer lines, and middle small transfer line is the example enrichment pipeline, example enrichment rear end of pipeline mouth connects sample pool, and other two small transfer lines of the tail end of described separating pipe are positioned at the both sides of example enrichment pipeline and link to each other with waste liquid pool; Described two groups of clamping plate type electrodes, wherein one group of arrangement of electrodes is in the bottom of separating pipe, realize the continuous separation of cell mixing sample, another group arrangement of electrodes is in the bottom of enrichment pipeline, realize the continuous enrichment of certain cell sample, sample in the sample inlet pool enters into separating pipe with buffered soln in the buffered soln pond after sample channel mixes, separation and the enrichment that dielectrophoresis separates the realization sample takes place according to the dielectric characteristics of self in sample component under the effect of the inhomogeneous field that two groups of clamping plate type electrodes produce, isolating different samples flow into sample pool and waste liquid pool respectively.
2. according to the described combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip of claim 1, it is characterized in that:
The described one group of clamping plate type electrode that is arranged in the bottom of separating pipe, being parallel to the axial plane electrode of separating pipe by three forms, target equates with the spacing of two lateral electrodes, spacing between the duct wall of described three electrodes and separating pipe equates, described two lateral electrodes are electrically connected, and are homopolarity, and different with described target electric polarity, described two lateral electrodes link to each other with the positive and negative electrode of power supply by lead respectively with target, form the separated region pilot circuit;
Another group clamping plate type electrode of the described bottom that is arranged in the enrichment pipeline becomes 90 ° and mutual disjunct plane clamping plate electrode to form by two, and described two plane clamping plate electrodes are connected to the positive and negative electrode of power supply, formation rich region pilot circuit through lead; Described two groups of clamping plate type electrodes all are the electrogilding membrane electrodes at glass substrate surface deposition 1～500nm.
3. combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip according to claim 2 is characterized by:
The described one group of clamping plate type electrode that is arranged in the bottom of separating pipe: the length of its two lateral electrode is 500～2000 μ m, and width is 5～20 μ m; The length of target is identical with two lateral electrodes, and the width of target is 30～55 μ m, and the spacing between target and two lateral electrodes is 1～20 μ m; Described separating pipe length is greater than the length of its bottom clamping plate type electrode, and the length of separating pipe is 800～2200 μ m, and the separating pipe width is 42～160 μ m;
Described another group clamping plate type electrode of bottom that is arranged in the enrichment pipeline: this group electrode width is 5～20 μ m, two electrodes of this group and duct wall angle at 45, electrode length is 20～28 μ m, and two electrode minimum spacings of this group are 5～10 μ m, and maximum spacing is 45～55 μ m; Described enrichment duct width is 45～55 μ m, and length is not shorter than 300 μ m;
The sample channel of described chip, separating pipe, enrichment pipeline overall depth are 10～50 μ m, and the thickness of described electrode is less than 1 μ m.
4. according to any described combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip of claim 1-3, it is characterized by: the substrate of described chip adopts glass or quartz material, adopt the formpiston of described three pipelines of Resins, epoxy photoresist material SU-8 material preparation, adopt polydimethylsiloxanepolymer polymer directly to pour into a mould reverse mould again, form three microchannel networks, then, dry up by cleaning and nitrogen, directly, form the combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip with substrate and the polydimethylsiloxanepolymer polymer cover plate bonding that contains microchannel.
5. combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip according to claim 2, it is characterized by: described separated region pilot circuit is connected by a two-way switch with the rich region pilot circuit, when two-way switch was connected with the pilot circuit of separated region, the rich region electrode kept trip condition; When switch is connected with the rich region pilot circuit, the electrode of separated region keeps trip condition, different two pilot circuits of ordered pair apply the high frequency low voltage when given, at the sample separation initial stage, two-way switch is communicated with the formation loop with power supply and the described one group of clamping plate type electrode that is arranged in the bottom of separating pipe, realizes the sample separation process; Then, two-way switch is turned to, another group clamping plate type electrode of power supply and the described bottom that is arranged in the enrichment pipeline is communicated with forms the loop, make sample after the separation in clamping plate central part realization enrichment.
6. combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chip according to claim 5 is characterized by: described voltage of supply scope is 0～20V, and frequency is 100KHz～20MHz.
7. according to claim 5 or 6 described combined splint microelectrode type micro-fluidic dielectrophoresis cell separation and enrichment chips, it is characterized by: in the enrichment pipeline, under the effect of rich region pilot circuit, the further enrichment of sample is embarked on journey, the final sample pool that arrives flows forward.
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