CN103084225B - High throughput microgel fixing method and special micro-fluidic chip thereof - Google Patents
High throughput microgel fixing method and special micro-fluidic chip thereof Download PDFInfo
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- CN103084225B CN103084225B CN201110331252.9A CN201110331252A CN103084225B CN 103084225 B CN103084225 B CN 103084225B CN 201110331252 A CN201110331252 A CN 201110331252A CN 103084225 B CN103084225 B CN 103084225B
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Abstract
The invention provides a high throughput microgel fixing method and a special micro-fluidic chip thereof. The micro-fluidic chip has a bilayer structure divided into an upper layer and a lower layer, wherein the upper layer is a droplet capture detector array, and the lower layer is a T-type droplet generation area and a droplet transportation channel. High throughput microgel is fixed by using a surface tension principle based on the chip. The micro-fluidic chip is simple in manufacture, convenient for experimental principle operations and stable in fixing effect, and can be easily combined flexibly and integrated on a large scale with other functional units on the chip. The micro droplets produced by the method can enter into the droplet capture array automatically under the drive of an injection pump and can be captured by a cylindrical microdroplet capture device successively.
Description
Technical field
The present invention relates to the online fixation in situ technology of microgel in micro-fluidic chip, specifically provide a kind of high flux microgel fixing means and micro-fluidic chip special thereof.
Background technology
In prior art, micro-fluidic chip (microfluidic chip) is also known as chip lab (lab on a chip), refer to integrated for the basic operation unit such as sample preparation, reaction, separation, detection involved in the fields such as biological and chemical or be substantially integrated on the chip of a piece several square centimeters (even less), network is formed by microchannel, whole system is run through, in order to replace a kind of technology of the various functions of standard biologic or chemical laboratory with controlled fluid.Recently, the microflow control technique based on drop causes increasing concern.Compared with microchannel, microlayer model has following advantage: size is less, and flux is higher, and mass-and heat-transfer is more rapid, transports and fixes more flexible, and can form a relatively stable controlled drop microenvironment; Therefore, in many high throughput analysis in chemistry and biology field, microlayer model is used to the screening (as protein crystal, DNA analysis etc.) of reaction condition as reactor, or is used for wrapping up various particle (comprising cell, bacterium etc.) to carry out various biological study.Polymer-network method is the spherical system that a class has cancellated intramolecular crosslinking, and its size is in micro-, nanometer scale.The character such as the size of microgel, internal structure and the kind of functional group that comprises can control simply by changing monomer composition, the consumption of crosslinking agent and preparation condition etc.Under the effect of dispersion stabilizer, microgel can disperse preferably in water or organic media and corresponding physics, chemical property are all closely related with its conformation change.For the change of external environmental condition as temperature, pH, ionic strength, electric field or magnetic field etc., namely microgel shows corresponding stimuli responsive characteristic.Macromolecule is synthesized as compared with dendrimer, block copolymer etc. with other, polymer-network method not only preparation method simply, easily introduces reactive group, and by selecting suitable polymerization, the size of microgel can be able to be effectively controlled from nanoscale to micron order.The special construction that polymer-network method has and special performance, it is made in the fields such as industrial production coating and paint, to obtain technical application, and have great application potential in drug controlled release, catalyst carrier, water body purification, Chemical Decomposition etc., and prepare inorganic research work that is micro-, nano material based on polymer-network method and also receive concern.Formed based on drop technique and solidification microgel can be quick, simply, stable operation also can accurately control the size of gel micro-ball by high flux, will be in conjunction with micro-fluidic chip, can be implemented as the in-situ solidifying of gel.
Therefore, people expect that obtaining one meets instructions for use and technique effect good high flux microlayer model fixing means and micro-fluidic chip special thereof.
Summary of the invention
A kind of high flux microgel fixing means and micro-fluidic chip special thereof are the object of this invention is to provide, the micro-fluidic chip manufacture related to is simple, convenient experimental operation, fixed effect is stablized, and be easy to carry out flexible combination with other functional units on chip and scale integrated.
The invention provides a kind of micro-fluidic chip, described micro-fluidic chip is double-decker, be divided into upper and lower two-layer, upper strata is drop catcher array, every 20 drop catchers by thin expanding channels together, each drop catcher is by drop transport channel connection simultaneously, and is all arranged in the top of drop transport passage; Lower floor is T-shaped drop formation district and drop transport passage, and a continuous phase entrance and four decentralized photo entrances are connected to form the sample introduction end in T-shaped drop formation district, and drop transport Tandem is after T-shaped drop formation district.
Micro-fluidic chip provided by the invention, the planform of described drop catcher is cylinder, and described drop catcher adds up to 80, and every 20 drop catchers are one group is arranged in continuously in drop transport passage.
Present invention also offers a kind of high flux microgel fixing means based on described micro-fluidic chip above, based on surface tension principle, concrete grammar is: the continuous phase on micro-fluidic chip be connected with two syringe pumps respectively by teflon pipe with decentralized photo entrance, under syringe pump drives, continuous phase and decentralized photo are injected chip respectively, forms continuous print monodispersity microlayer model in T-shaped drop formation district afterwards; The microlayer model of continuous generation continues flow and enter drop catcher array under syringe pump drives, drop when flowing through grabber down channel because capillary acting in conjunction upwards enters grabber; Subsequent droplet flows through from the down channel of the drop catcher successfully fixing drop, then be sequentially trapped in follow-up drop catcher, when microlayer model all enters in drop catcher, stop syringe pump, keep 30 minutes under whole chip apparatus being placed in the condition of 4 degrees Celsius, drop is solidified into gel, forms high-throughout microgel array.
High flux microgel fixing means provided by the invention, described continuous phase is oil phase (preferably fluoridizing liquid-fluorine carbon-40(FC-40)), decentralized photo is agarose solution (being preferably 0.5% agarose solution).
High flux microgel fixing means provided by the invention, the curing of described microgel is by reducing temperature mediation, and temperature is reduced to 4 degree by room temperature.
Tool of the present invention has the following advantages:
1) microlayer model that the present invention produces can automatically enter droplet capture array and is sequentially trapped in cylindrical microlayer model grabber under syringe pump drives;
2) creativeness of the present invention is on chip, utilize capillary effect to complete the fixing of gel, and utilize temperature to mediate microgel solidification, chip design is simple, and mode of operation is easy, and capture effect is stablized;
3) this invention simplifies gel fixation principle and process, be easy to carry out flexible combination with other functional units and scale integrated.
Accompanying drawing explanation
Fig. 1 is integrated micro-flow control chip platform and integrally structural representation, and wherein: 1 is continuous phase entrance, 2 is decentralized photo entrance, and 3 is capture gel array, and 4 is waste liquid outlet, and this chip comprises the entrance of 4 decentralized photos, can enter 4 kinds of different samples simultaneously;
Fig. 2 is the enlarged drawing of T-shaped passage and a capture gel device in capture gel array, and wherein, 1 is the forerunner's microlayer model formed, and 2 is a microgel grabber;
Fig. 3 is the fixation procedure schematic diagram of microlayer model in capture array, wherein: A illustrates that forerunner's drop 1 is positioned at lower floor's transport passage, because space constraint is strip; B illustrates that forerunner's drop 1 flow to below grabber, because capillary effect is to rising, starts to enter in the diameter grabber larger than transport passage; C illustrates that forerunner drop 1 continues to rise, and enters grabber completely, and the minimum spheroidal state of recovery table surface tension and being caught by stable, forerunner's drop 2 continues flow forward; D illustrates that forerunner's drop 1 is captured in grabber by stable, and forerunner's drop 2 continues forward from the transport passage of lower floor, does not merge, enter next capturing unit with forerunner's drop 1; E illustrates that forerunner's microlayer model 1 is placed 30 minutes after coagulations at 4 degrees Celsius and become microgel 3;
Fig. 4 is the online capture array pictorial diagram of microgel;
Fig. 5 be ACCM cell in agarose microgel 1,2, the proliferative conditions pictorial diagram of 3 days;
Fig. 6 be ACCM cell in agarose microgel 1,2, the proliferative conditions statistical chart of 3 days.
Detailed description of the invention
The following examples will be further described the present invention, but not thereby limiting the invention.
Embodiment 1 one kinds is based on high flux microgel fixing means and special chip thereof on the micro-fluidic chip of surface tension principle.
For the micro-fluidic chip of high flux microgel fixing means, this chip is double-decker, be divided into upper and lower two-layer, upper strata is drop catcher array, every 20 drop catchers by thin expanding channels together, each drop catcher is by drop transport channel connection simultaneously, and is all arranged in the top of drop transport passage; Lower floor is T-shaped drop formation district and drop transport passage, and a continuous phase entrance and four decentralized photo entrances are connected to form the sample introduction end in T-shaped drop formation district, drop transport Tandem after T-shaped drop formation district, as shown in Figure 1.
A kind of based on high flux microgel fixing means on the micro-fluidic chip of surface tension principle, method detailed process is: the continuous phase (fluoridizing liquid-fluorine carbon-40(FC-40) by chip) be connected with syringe pump respectively by teflon pipe with decentralized photo (agarose solution of 0.5%W/V) entrance, under syringe pump drives, continuous phase and decentralized photo are injected chip respectively, generates district at T-shaped microlayer model afterwards and form continuous print monodispersity microlayer model; The microlayer model of continuous generation continues flow and enter drop catcher array under syringe pump drives, drop when flowing through grabber down channel because capillary effect upwards enters cylinder grabber, subsequent droplet then from below passage flow through, be then sequentially trapped in cylindrical drop catcher; After microlayer model is caught completely, close syringe pump to stop drop producing and fluid flowing.The environment chip of catching drop being placed in 4 degrees Celsius keeps 30 minutes, and microlayer model is solidified into microgel, and process as shown in Figure 3.
Described microlayer model catches principle: generate at T-shaped microlayer model the microlayer model that district produces and continue flow and enter drop catcher array under syringe pump drives, drop when flowing through grabber down channel because capillary effect upwards enters cylinder grabber; Drop is in squeezed state in down channel, and recover spherical after entering grabber, surface tension is minimum, and thus state is firm; Subsequent droplet then from below passage flow through, then by the cylindrical drop catcher that is sequentially trapped in below, every 20 cylindrical drop catchers, by narrow expanding channels, have the effect of shunting, better can improve capture rate to continuous phase.
Embodiment 2 one kinds is based on the online fixing means of the multi-component microgel of high flux on the micro-fluidic chip of surface tension principle
Concrete grammar is: be connected with four syringe pumps respectively by teflon pipe at the different decentralized photo entrances of chip, under syringe pump drives, continuous phase and decentralized photo is injected chip respectively, generates district afterwards form continuous print monodispersity microlayer model at T-shaped microlayer model; Decentralized photo is respectively the agarose solution of different colours ink markings 0.5%W/V.The microlayer model of continuous generation continues flow and enter drop catcher array under syringe pump drives, drop when flowing through grabber down channel because capillary effect upwards enters cylinder grabber, after microlayer model is caught completely, close syringe pump to stop drop producing and fluid flowing.The environment chip of catching drop being placed in 4 degrees Celsius keeps 30 minutes, and microlayer model is solidified into microgel, forms multi-component agarose microgel array, as shown in Figure 4.
Embodiment 3 one kinds of online fixing means of high flux microgel are used for the dimensional culture of cell.
Concrete grammar is: the continuous phase on chip be connected with two syringe pumps respectively by teflon pipe with decentralized photo entrance, under syringe pump drives, continuous phase and decentralized photo are injected chip respectively, afterwards generate at T-shaped microlayer model the microlayer model that district forms continuous print monodispersity parcel cell, continuous phase is FC-40, and decentralized photo is the agarose ACCM cell suspension of 0.5%W/V.The microlayer model of the parcel cell of continuous generation continues flow and enter drop catcher array under syringe pump drives, drop when flowing through grabber down channel owing to upwards entering cylinder grabber with capillary acting in conjunction; Subsequent droplet flows through from the down channel of the drop catcher successfully fixing drop, is then sequentially trapped in follow-up cylindrical drop catcher.Keep 30 minutes under the chip of agarose microarray of parcel cell is placed in 4 degrees celsius, the microlayer model of parcel cell is solidified into microgel, then connect cell culture fluid at continuous phase entrance by syringe pump, put into three days (as shown in Figure 5) of cell culture incubator Continuous Cultivation.As can be seen from the figure the proliferative conditions of ACCM cell in agarose microgel, can find out that the propagation of the cell of ACCM in the three dimensional matrix of agarose is not clearly (growth becomes as shown in Figure 6) by icon, this is the rule of proliferation meeting three-dimensional cell cultivation, cultivate in agarose microgel at ACCM cell and dye with PI for the 3rd day, the survival rate of statistics cell is 75%, and growing state is good.
Claims (2)
1. a micro-fluidic chip, is characterized in that: described micro-fluidic chip is double-decker, is divided into upper and lower two-layer,
Upper strata is drop catcher array, and by thin expanding channels together, each drop catcher is by drop transport channel connection simultaneously, and is all arranged in the top of drop transport passage for every 20 drop catchers;
Lower floor is T-shaped drop formation district and drop transport passage, and a continuous phase entrance and four decentralized photo entrances are connected to form the sample introduction end in T-shaped drop formation district, and drop transport Tandem is after T-shaped drop formation district;
The planform of described drop catcher is cylinder;
Described drop catcher adds up to 80, and every 20 drop catchers are one group is arranged in continuously in drop transport passage.
2. the high flux microgel fixing means based on micro-fluidic chip described in claim 1, based on surface tension principle, it is characterized in that: the continuous phase on micro-fluidic chip is connected with two syringe pumps by teflon pipe respectively with decentralized photo entrance, under syringe pump drives, continuous phase and decentralized photo are injected chip respectively, forms continuous print monodispersity microlayer model in T-shaped drop formation district afterwards;
The microlayer model of continuous generation continues flow and enter drop catcher array under syringe pump drives, drop when flowing through grabber down channel because capillary acting in conjunction upwards enters grabber; Subsequent droplet flows through from the down channel of the drop catcher successfully fixing drop, then be sequentially trapped in follow-up drop catcher, the chip capturing drop is put in 4 degree of environment and keeps 30 minutes, drop is solidified into gel, forms high-throughout microgel array;
Described continuous phase is oil phase, and decentralized photo is agarose solution; Described continuous phase is for fluoridizing liquid-fluorine carbon-40, and decentralized photo is the agarose solution of 0.5%;
The curing of described microgel is by reducing temperature mediation, and temperature is reduced to 4 degree by room temperature.
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