WO2014082377A1

WO2014082377A1 - Microfluidic chip

Info

Publication number: WO2014082377A1
Application number: PCT/CN2013/001451
Authority: WO
Inventors: 陈立桅; 甘明哲; 汤云芳
Original assignee: 中国科学院苏州纳米技术与纳米仿生研究所
Priority date: 2012-11-28
Filing date: 2013-11-27
Publication date: 2014-06-05

Abstract

A microfluidic solution concentration generation chip of the present invention can obtain a specific concentration gradient as required, thus satisfying various concentration gradient requirements, and achieve not only the suspension cultures of microorganisms or cells but also the detection of specific compositions in culture solutions. The microfluidic chip of the present invention realizes separation between liquid flow pipes, has a lower requirement for precision of assembly of various layers, and can arrange the liquid flow pipes much closer. The gas concentration in the microfluidic chip of the present invention is generated depending on different amounts of a gas diffusing in the solution through a gas-permeable membrane at multiple crossover points or in different crossover areas; the concentration of the dissolved gas mainly relies on the number of crossover points or the size of the crossover area; the present invention avoids the use of a pyramid-shaped gas mixing and distribution structure, thus saving chip area, and improving the integration of culture units in the chip.

Description

Microfluidic Chip

[Technical Field]

The invention belongs to the technical field of micromachining, and in particular relates to a microfluidic chip. 【Background technique】

Noo Li Jeon, in patent WO200222264, describes a pyramidal microfluidic chip capable of rapidly forming a concentration gradient. The chip has three solution inlets, respectively injected into a low concentration, medium concentration and high concentration solution, through a 9-stage branch pipeline network. After the mixing was dispensed, the mixed solution was discharged from the nine outlets, and the concentration of each of the outlet solutions formed a gradient. By inputting the chip with a smaller amount of the original concentration solution, various concentration gradients can be obtained, and the whole process is miniaturized and automated, and the steps of repeated addition of the solution in the conventional operation are avoided, which can save manpower and improve operation efficiency. However, the chip design relies on a multi-stage branch pipe network. As the number of concentration outlets increases, the number of branch pipe network stages increases accordingly, which takes up a large chip area, which is not conducive to the integration of other chip functional units in the later stage. The number of stages also increases the injection pressure, which increases the difficulty of controlling the injection flow rate. The concentration is greatly affected by the injection flow rate, and the flow rate needs to be precisely controlled. In addition, the type of solution gradient generated by a single set of pyramidal gradient generating units is also limited. Although several kinds of gradient types can be added by combining multiple gradient elements, the types are still limited, and it is difficult to meet the requirements of diverse concentration gradient experiments. Will occupy more chip area.

Jian Liu describes a microfluidic reaction array based on cyclic mixing in the patent US 2010/0104477. The array consists of 400 square closed liquid mixing units and fluid control structures such as microvalves and micropumps. For each liquid mixing unit, different liquids can be injected into the pipe of one side or two sides of the square unit by control, and then the square closed units are separated from each other by a micro valve, and the micro pump is started to carry out the solution mixing in the unit. The chip can realize the injection and mixing of different liquid batches, has high operation efficiency and occupies a small chip area, and provides a new idea for realizing multi-unit batch injection and rapid mixing on a chip. However, the structure of each unit in the reaction array is the same, and the concentration of the solution produced by mixing each unit is the same, and it cannot be directly used for batch production to produce a plurality of solution concentrations, which is not directly

) - people Used for a variety of concentration gradient experiments.

In 2002, Todd Thorsen, et al. reported a microfluidic optical comparison chip (Science 298, 580 (2002); DOI: 10.1 126/science. 10769%). The chip contains 256 reaction units, each unit consisting of a chamber containing bacteria and a chamber containing a color developing solution with a microvalve between the two chambers. After the injection is completed, the microvalve is opened to allow the bacteria to react with the color developing solution, and the generated fluorescent signal can be used to determine whether the bacteria express a specific protein. Although the detection flux is very high, the cell can be proliferated due to the inability to carry out suspension culture in the chip, and is only used for screening individual bacteria, and cannot be used for diverse microbial screening targets, and the versatility is not high.

In 2005 Nicolas Szita reported a multichannel microfluidic microreaction chip (DOI: 10.1039/b504243g). The chip integrates four reaction chambers, each with a micro-stirring paddle for microbial suspension culture, and two patch sensors for detection of pH and dissolved oxygen content in the culture medium. Although the chip can realize microbial suspension culture and detect the pH and dissolved oxygen concentration in the culture solution, due to the large volume of a single reaction chamber, about several hundred microliters, it is necessary to process a micro-mixing paddle, integrated patch sensor, and manufacturing process. Complex, it is difficult to significantly increase the number of chip units.

A microfluidic cell suspension culture chip is disclosed in the Chinese Patent Nos. CN201 1 10316751.0 and CN201 1 10142095.7, respectively, and the chips mentioned in the two patents can realize batch parallel suspension culture of hundreds of channels of microorganisms. However, the chip can only determine the number of cells in the culture solution by cell counting, lacks the culture liquid detection structure, and cannot measure the concentration of a specific component in the culture solution.

A cell culture that produces a gradient of dissolved oxygen concentration is described in the literature (Joe F. Lo Elly Sinkala, David T. Eddington. Oxygen gradients for open well cellular cultures via microfluidic substrates. Lab on a Chip, 2010, 10 2394 - 2401 ). Microfluidic chip, the upper layer of the chip has a cylindrical static culture chamber for cell culture, a gas permeable membrane in the middle, and a gas pipeline network in the lower layer. There are two ways for the gas pipeline network to generate the oxygen concentration gradient. One is to set up two horizontally parallel main pipes. There are a plurality of longitudinal parallel pipes between the two pipes. These pipes do not cross the main pipes and pass through the two main pipes. With nitrogen and oxygen, the gas will diffuse into the longitudinal parallel pipe and form an oxygen concentration gradient; another way of generating oxygen is Nitrogen and oxygen are simultaneously introduced into the pyramidal branch network channel for mixing, and an oxygen concentration gradient is formed at several outlets. Gases containing different oxygen concentrations diffuse through the gas permeable membrane into the culture chamber of the upper layer, thereby changing the dissolved oxygen concentration in different regions of the same culture chamber. The chip is suitable for studying the effects of various dissolved oxygen concentrations on cell culture. However, the culture chamber of the chip is still large, and it is difficult to further increase the number of culture chambers on the chip. In addition, the chip is used for culture in which the cells are left to stand, and is not suitable for use in a microbial suspension culture environment.

The literature (Raymond HW Lam, Min-Cheol Kim, Todd Thorsen. Culturing aerobic and anaerobic bacteria and mammalian cells with a microfluidic differential oxygenator. Analytical chemistry, 2009, 81, 5918-5924) describes a gradient of dissolved oxygen concentration The cell culture chip, in which the oxygen concentration gradient is filled with nitrogen and oxygen into the pyramidal branch pipe network, after multiple mixing, produces an oxygen concentration gradient at the outlet. Gases of different oxygen contents enter the lower cell culture channels through diffusion through the membrane to form different dissolved oxygen conditions. In the chip, the gas conduit is parallel to the cell culture conduit, and the cells are infused in the culture pipeline and then infused with gas to produce different oxygen concentrations for static culture. However, since the gas pipe and the culture pipe are stacked in parallel, the difficulty of aligning the package between the upper and lower layers of the chip is increased, and in particular, when there are a large number of culture pipes, precise alignment is more difficult. Uncertainty in the variation of the horizontal distance between the gas pipeline and the culture pipeline also changes the gas diffusion distance, thereby affecting the dissolved oxygen content in the pipeline. In addition, the chip is still based on a cell-based culture mode and is not suitable for suspension culture of microorganisms.

As shown in FIG. 12, in the prior art, a microfluidic chip includes a stacked culture layer, an elastic diaphragm layer and a driving layer. The elastic diaphragm layer is located between the culture layer and the driving layer, and a plurality of liquids are distributed on the culture layer. The flow pipe 1 1 is connected to the liquid injection pipe 12 via the liquid flow pipe 11. The drive layer is provided with a drive channel 13 which is an interdigitated pneumatic microvalve for achieving separation between adjacent flow conduits 1 1 . In this technique, the overlap between the drive layer and the culture layer needs to be accurately positioned in two dimensions of the horizontal plane. The ideal positioning of the interdigitated pneumatic microvalve is: In the transverse direction, two adjacent interdigitated microvalves are respectively located on both sides of the liquid flow pipe 1 1 , closely arranged but not overlapping with the liquid flow pipe 1 1 , longitudinally, the fork The finger-shaped pneumatic microvalve should cross the liquid injection channel 12, and the length of the interdigital finger should be as short as possible to reduce the occupied core. Area, improve integration. However, there are errors in the alignment of the two layers of the chip in the two dimensions of the horizontal plane during the chip fabrication process. In order to prevent the error caused by the processing from affecting the use of the chip, it is necessary to increase the pitch of the interdigital pneumatic valve and the length of the interdigital finger, which inevitably increases the chip area occupied by the micro valve and reduces the integration degree of the chip. In addition, when the two-layer chip pattern slightly differs due to the difference in substrate shrinkage, even if it is precisely aligned in a certain area, the dimensional deviation between the two layers increases as the distance of the alignment point increases, resulting in The volume of each reaction chamber is inconsistent, affecting the accuracy of detection, and even some units are completely unusable for detection.

[Summary of the Invention]

It is an object of the present invention to provide a microfluidic chip to solve the problems in the prior art. In order to solve the above technical problem, the present invention adopts the following technical solutions:

The invention discloses a microfluidic solution concentration generating chip, which comprises a stacked culture layer and a driving layer, wherein a plurality of culture units are arranged side by side on the culture layer, and a first driving valve is distributed on the driving layer. The first drive valve forms an intersection with the culture unit at a first position that divides the culture unit into an upper culture unit and a lower culture unit, and at least a portion of the lower culture unit of the culture unit has a different volume.

Preferably, in the above microfluidic solution concentration generating chip, the culture unit is a circulation channel connected end to end, and the driving layer is provided with a circulation driving valve, and the circulation driving gate forms an intersection with the culture unit. And driving the liquid circulation flow in the culture unit.

Preferably, in the above microfluidic solution concentration generating chip, a liquid flow conduit is connected between the culture units, and a connection point between the liquid flow conduit and the culture unit is close to the first position.

Preferably, in the above microfluidic solution concentration generating chip, the driving layer is further provided with a second driving valve, and the second driving valve controls the conduction or the cutoff of the liquid flow conduit between the adjacent culture units.

Preferably, in the above microfluidic solution concentration generating chip, the first driving valve is parallel to the liquid flow conduit.

Preferably, in the microfluidic solution concentration generating chip described above, the first driving is broadly linear or stepped. The invention also discloses a microfluidic culture detection chip, comprising a stacked culture layer and a driving layer, wherein the culture layer is distributed with at least one culture detecting unit, and each culture detecting unit comprises a culture channel connected end to end and a detection channel communicating with the culture channel, wherein the drive layer is distributed with a circulation drive pump and a detection drive valve, wherein the circulation drive pump forms an intersection with the culture channel and drives the culture channel The culture fluid in the channel circulates; the detection drive valve includes at least two drive valves and each intersects the detection channel, and controls the conduction or the turn-off of the detection channel at the intersection.

Preferably, in the above microfluidic culture detecting chip, the culture detecting unit is further connected with a color developing liquid injection channel, and the detecting driving valve is located between the circulating driving pump and the color developing liquid injection channel.

Preferably, in the above microfluidic culture detecting chip, a third driving valve is further disposed on the driving layer, and the third driving valve respectively intersects the color developing liquid injection channel and the detecting channel to simultaneously control the device. The conduction of the color liquid injection channel and the detection channel is described.

Preferably, in the above microfluidic culture detecting chip, the third driving valve is linear. Preferably, in the above microfluidic culture detection chip, a fifth driving valve is further disposed on the driving layer, and the fifth driving valve controls conduction of the coloring liquid injection channel between adjacent culture detecting units. cutoff.

Preferably, in the above microfluidic culture detection chip, a fourth driving valve is further disposed on the driving layer, and the fourth driving valve is located above the culture channel and intersects with the culture channel. To control the conduction and the cutoff of the culture channel at the intersection, the fourth drive valve is located between the cycle drive pump and the detection drive width.

Preferably, in the above microfluidic culture detecting chip, the culture detecting unit is further connected with a cleaning liquid injection channel, and the cleaning liquid injection channel is located between the fourth driving valve and the detecting channel.

Preferably, in the above microfluidic culture detecting chip, the cleaning liquid injection channel is in communication with a junction of the culture channel and the detection channel.

Preferably, in the above microfluidic culture detection chip, the sixth layer is further distributed on the driving layer. The drive valve controls the conduction or the cut-off of the cleaning liquid injection passage between the adjacent culture detecting units.

Preferably, in the above microfluidic culture detection chip, a ninth driving valve is further disposed on the driving layer, and the ninth driving valve and the culture channel form an intersection at a first position, the first position The culture channel is divided into an upper culture unit and a lower culture unit.

Preferably, in the above microfluidic culture detection chip, at least a part of the culture unit has a different volume of the lower culture unit.

Preferably, in the above microfluidic culture detecting chip, a liquid flow conduit is connected between the culture channels, and a connection point between the liquid flow conduit and the culture channel is close to the first position.

Preferably, in the above microfluidic culture detecting chip, the driving layer is further provided with a tenth driving valve, and the tenth driving valve controls the conduction or the cutoff of the liquid flow conduit between the adjacent culture channels.

Preferably, in the above microfluidic culture detecting chip, the ninth driving valve is parallel to the liquid flow conduit.

Preferably, in the above microfluidic culture detecting chip, the ninth driving valve is linear or stepped.

The invention also discloses a microfluidic chip, comprising a stacked culture layer and a driving layer, wherein the culture layer is distributed with a liquid flow pipe and a liquid injection channel, and the liquid injection channel is connected to the liquid flow pipe. A drive valve is disposed on the drive layer, and the drive valve respectively intersects the liquid flow conduit and the liquid injection passage to simultaneously control conduction and cutoff of the liquid flow conduit and the liquid injection passage.

Preferably, in the above microfluidic chip, the driving valve is linear.

Preferably, in the above microfluidic chip, the driving valve forms at least two intersections with the liquid injection channel.

The invention also discloses a microfluidic culture chip, comprising:

a culture layer in which at least one culture unit is distributed;

a pneumatic control layer, wherein the pneumatic control layer is distributed with a gas supply pipe, and the gas supply pipe is closed with the element; An elastic gas permeable membrane is formed between the culture layer and the pneumatic control layer, and the gas in the gas supply conduit enters the solution in the culture unit through the elastic gas permeable membrane at the intersection.

Preferably, in the above microfluidic culture chip, the elastic gas permeable membrane constitutes one side wall of the gas supply pipe.

Preferably, in the above microfluidic culture chip, the elastic gas permeable membrane constitutes one side wall of the culture unit.

Preferably, in the above microfluidic culture chip, the culture layer, the pneumatic control layer and the elastic gas permeable membrane are each made of a gas permeable material.

Preferably, in the above microfluidic culture chip, the gas permeable material is polydiphenylsiloxane.

Preferably, in the microfluidic culture chip described above, at least a portion of the side wall of the gas supply conduit is constituted by the pneumatic control layer.

Preferably, in the above microfluidic culture chip, at least a part of the side wall of the culture unit is constituted by the culture layer.

Preferably, in the above microfluidic culture chip, the culture unit comprises an annular closed conduit, and a circulation drive pump is further distributed in the pneumatic control layer, and the circulation drive pump forms an intersection with the culture unit and drives The liquid in the culture unit circulates.

Preferably, in the above microfluidic culture chip, at least a first culture unit and a second culture unit are distributed in the culture layer, and the gas supply conduit intersects with the first culture unit and the second culture unit. The number of times is different from the Z or cross area.

Preferably, in the microfluidic culture chip described above, the gas in the gas supply conduit is selected from the group consisting of oxygen, carbon dioxide or ammonia.

The advantages of the present invention over the prior art are:

1. The microfluidic solution concentration generating chip of the present invention is capable of rapidly generating a plurality of solution concentrations and performing microbial culture therein. The chip does not need to be added to the pyramidal branch pipeline network. The gradient generating unit has a small footprint and is easy to implement arraying. It does not require precise flow rate control or long-term balance. The injection is simple, and a specific gradient concentration can be obtained according to requirements, which can meet various gradient concentrations. demand. 2. The microfluidic culture detection chip of the invention integrates the suspension culture channel and the detection channel on the same chip, and simultaneously realizes suspension culture and detection of microorganisms; meanwhile, the detection drive includes at least two drive valves, which can be realized The culture solution was tested multiple times in different time periods.

3. The microfluidic solution concentration of the present invention and the culture detection chip integrate the concentration generation, the suspension culture channel and the detection channel on the same chip, and can obtain a specific gradient concentration according to requirements, which can meet various gradient concentration requirements. The gradient generating unit has a small occupied area, is easy to realize arraying, does not require precise flow rate control or long-term balance, and is capable of injecting a single cylinder; and simultaneously realizes suspension culture and detection of microorganisms; in addition, the detection driving valve includes at least two driving valves, It is possible to carry out multiple tests on the culture medium in different time periods.

4. The invention crosses the liquid flow pipe and the liquid injection channel by driving the wide, not only can realize the function of separating the liquid flow pipes, but also because the driving valve is linear, the assembly precision of each layer is lower, and the upper and lower layers are stacked. The combination only needs to be parallel in one dimension, and it is not necessary to precisely align the layers in two dimensions of the horizontal plane, which reduces the difficulty of chip fabrication and helps to increase the number of liquid flow pipelines.

5. The gas concentration in the microfluidic chip of the present invention is formed by the fact that the gas diffuses into the solution through the gas permeable membrane under a plurality of intersections or different cross-sectional areas, and the concentration of the dissolved gas is more important. The number of intersections and the size of the intersections avoid the use of a pyramid-shaped gas mixing distribution structure, which saves the chip area and improves the integration of the culture units in the chip. At the same time, there is no need to precisely control the inlet gas flow, just need to pass, and the operation is simpler. In the process of chip fabrication, it is easier to cross-ply the pneumatic control pipe and the liquid flow pipe to reduce the difficulty of making the chip. At the same time, the technical solution of the present invention can carry out microbial suspension culture while controlling the dissolved oxygen concentration condition, and the variety and application range of the culture are further expanded as compared with the prior art only static culture.

[Description of the Drawings]

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings to be used in the embodiments or the description of the prior art will be briefly described below. Obviously, the drawings in the following description are only Some embodiments described in this application are for those of ordinary skill in the art In other words, other drawings can be obtained based on these drawings without paying creative labor. 1a is a plan view showing a microfluidic solution concentration generating chip in a first embodiment of the present invention; FIG. 1b is a plan view showing a culture layer in the first embodiment of the present invention;

Figure lc is a plan view showing the driving layer in the first embodiment of the present invention;

Figure Id is a cross-sectional view taken along line 1D in Figure 1a;

2 is a plan view showing a microfluidic solution concentration generating chip according to a second embodiment of the present invention; FIG. 3 is a plan view showing a microfluidic solution concentration generating chip according to a third embodiment of the present invention; FIG. 5a is a plan view of a microfluidic culture detection chip according to a fifth embodiment of the present invention; FIG. 5b is a cross-sectional view along line 1D of FIG. 5a;

Figure 6a is a plan view showing a microfluidic culture detecting chip in a sixth embodiment of the present invention; and Figure 6b is a cross-sectional view taken along line 2D in Figure 6a;

7 is a top plan view showing a microfluidic solution concentration occurrence and culture detecting chip in a seventh embodiment of the present invention;

Figure 8 is a cross-sectional view taken along line 1D of Figure 7;

Figure 9 is a plan view showing the microfluidic solution concentration generation and culture detecting chip in the eighth embodiment of the present invention;

Figure 10 is a plan view showing the microfluidic solution concentration generation and culture detecting chip in the ninth embodiment of the present invention;

Figure 1 is a top plan view showing the microfluidic solution concentration generation and culture detecting chip in the tenth embodiment of the present invention;

12 is a schematic structural view of a microfluidic chip in the prior art;

13 is a schematic structural view of a microfluidic chip according to an eleventh embodiment of the present invention (one liquid flow pipe);

14 is a schematic structural view of a microfluidic chip according to an eleventh embodiment of the present invention (a plurality of liquid flow pipes);

15 is a schematic structural view of a microfluidic chip according to a twelfth embodiment of the present invention; 16 is a schematic structural view of a microfluidic chip according to a thirteenth embodiment of the present invention;

Figure 17a is a schematic view showing the structure of a microfluidic culture chip in the fourteenth embodiment of the present invention; Figure 17b is a schematic view showing the structure of the culture layer in the fourteenth embodiment of the present invention;

Figure 17c is a schematic view showing the structure of the pneumatic control layer in the fourteenth embodiment of the present invention; Figure 1 7d is a cross-sectional structural view along line 1 D shown in Figure 17a;

FIG. 18 is a schematic structural view of a microfluidic culture chip according to a fifteenth embodiment of the present invention; FIG. 19 is a schematic structural view of a microfluidic culture chip according to a sixteenth embodiment of the present invention; FIG. 21 is a schematic structural view of a microfluidic culture chip according to an eighteenth embodiment of the present invention; FIG. 22 is a nineteenth embodiment of the present invention; Schematic diagram of the structure of the microfluidic culture chip in the example; FIG. 23 is a schematic structural view showing the case where the drive valve or the drive pump is a solenoid valve in the specific embodiment of the present invention;

Fig. 24 is a schematic view showing the structure of a driving valve or a driving pump which is a photo-deformation valve in a specific embodiment of the present invention.

【detailed description】

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the drawings. The embodiments of the invention shown in the drawings and described in the drawings are merely exemplary, and the invention is not limited to the embodiments.

In this context, it is also to be noted that in order to avoid obscuring the invention by unnecessary detail, only the structures and/or process steps closely related to the solution according to the invention are shown in the drawings, and the Other details that are not relevant to the present invention.

Additionally, repeated numbers or labels may be used in different embodiments. These repetitions are merely illustrative of the invention and are not meant to be any of the various embodiments and/or structures discussed.

Finally, it should also be noted that the terms "include", "include" or any other variant thereof It is intended to cover a non-exclusive inclusion, such that a process, method, article, or device that comprises a plurality of elements includes not only those elements but also other elements not specifically listed, or Or the elements inherent in the device.

1a to 1d are respectively a plan view of the microfluidic solution concentration generating chip, a plan view of the culture layer, a plan view and a cross-sectional view of the driving layer in the first embodiment of the present invention.

As shown in FIG. 1A to FIG. 1D, the microfluidic solution concentration generating chip 10 includes a culture layer 11 composed of at least one of a polymer, a hydrogel, a silicon wafer, a quartz, a glass, and a metal material. Alternatively or in combination, the culture layer 11 is made of polydimethylsiloxane.

A plurality of culture units 111 are arranged side by side on the culture layer 11, and the culture unit 111 is a unit having the same shape and volume, and the units are arranged in parallel, and the culture unit 111 is a circulation channel connected end to end.

The culture unit ill is connected with a liquid flow pipe 112. The liquid flow pipe 112 includes a plurality of liquid flow pipe units 1121, each of the liquid flow pipe units 1121 is connected between adjacent culture units 111, and the liquid flow pipe unit 1121 is laterally Extending and perpendicular to the culture unit 111, the plurality of liquid flow conduit units 1121 are arranged in a stepped manner.

The upper end of the culture unit 111 is in common communication with the liquid flow conduit 113, and the lower end is in common communication with the liquid flow conduit 114. The flow conduits 113 and 114 are in communication with the outside. The solution can be simultaneously injected into all the cultivation units 11 through the liquid flow conduit 114, and the liquid flow conduit 113 can serve as an outlet for the solution flow.

It is conceivable that all the culture units 111 can be separate units, that is, the upper end or the lower end of the culture unit 111 are not connected, and each has an independent outlet and an inlet, so that different solutions can be injected into different culture units in. .

The elastic layer 12 is laminated on the upper side of the culture layer 11, and the elastic diaphragm layer 12 is formed of an elastic polymer material. Preferably, the elastic diaphragm layer 12 is made of polydimethicone.

A driving layer 13 is formed above the elastic diaphragm layer 12, and the driving layer 13 is formed of at least a combination of any one or more of a polymer, a hydrogel, a silicon wafer, a quartz, a glass, and a metal material. The driving layer 13 is made of polydidecylsiloxane.

A circulation drive pump 131 is disposed on the drive layer 13, and the circulation drive pump 131 forms an intersection with the culture unit 111, and drives the liquid in the culture unit 111 to circulate. The circulation drive pump 131 is preferably a pipe whose both ends are connected to the outside. When high pressure gas is injected into the circulation drive pump 131, the elastic diaphragm layer 12 under the circulation drive pump 131 is bent downward to block the lower portion of the elastic diaphragm layer 12. The culture unit 111, when the high pressure gas is withdrawn, the elastic diaphragm layer 12 is restored, and the lower culture unit 111 is connected, which is a microvalve known in the art of microfluidics.

As shown in FIG. 23, the cyclic drive pump 131 may also be an electromagnet 200. The lower side of the culture unit 111 is provided with a metal substrate 300 that can be attracted to the electromagnet. The metal substrate is preferably an iron substrate. When the electromagnet is energized, The liquid in the culture unit 111 is blocked by the suction from the metal substrate. After the electromagnet is deenergized, the magnetic properties disappear and the channel of the culture unit 111 is turned on.

As shown in FIG. 24, in another embodiment, the driving layer 400 is made of a photo-deformation polymer material. When light of a specific intensity and wavelength is irradiated on a specific region 500 above the channel of the culture unit 111, The photodeformation material of the region is deformed, and the liquid in the culture unit 111 is blocked downward to block the liquid, and the liquid is cut off; when the light stops, the drive layer 400 is deformed.

It is conceivable that switching between conduction and blocking can also be achieved by providing a micro wide door in the channel of the culture unit.

It should be noted that the above-described solenoid valve mode of Fig. 23 and the photo-induced valve mode of Fig. 24 are equally applicable to the drive pump and the drive valve hereinafter, and therefore will not be described below.

The elastic diaphragm layer 12 may be a separate layer or a part of the driving layer 13 or the culture layer 11.

The circulation drive pump 131 is two or three parallel pipes, and by sequentially pressing at a specific timing, the liquid in the lower culture unit 111 can be squeezed to flow in one direction, which is a micro pump known in the art of t flow control.

The circulation drive structure formed by the circulation drive pump 131 and the culture unit 111 and the principle thereof are disclosed in the Chinese Patent Nos. CN201110316751.0 and CN201110142095.7, and the present embodiment will not be described again.

Also distributed on the drive layer 13 is a first drive valve 132 that intersects the culture unit 111 at a first position A that divides the culture unit 111 into an upper culture unit 1111 and a lower culture unit 1112. The first drive valve 132 includes a plurality of drive valve units 1321, each of which is wide The unit 1321 is located above the adjacent two branch channels of the adjacent culture unit 111, and the drive valve unit 1321 extends in the lateral direction and is perpendicular to the culture unit 111, and the plurality of drive valve units 1321 are arranged in a stepwise manner. The connection between the liquid flow pipe unit 1121 and the culture unit 111 is as close as possible to the first position

A.

A second driving valve 133 is further disposed on the driving layer 13, and the second driving valve 133 respectively intersects the liquid flow pipe unit 1121 between the adjacent culture units 111, and forms a micro valve at the intersection to control the liquid flow pipe. The unit 1121 is turned on or off. The second driving valve 133 also forms an intersection with the liquid flow conduit 114 and forms a microvalve at the intersection. When the high pressure gas is introduced into the second driving valve 133, the second driving valve 133 can close the opening of the lower end of the culture unit 11. At the same time, the separation between the lower ends of the culture unit 111 is achieved.

The upper and lower ends of the drive layer 13 are also distributed with a third drive valve 134 and a fourth drive valve 135, respectively. The third driving valve 134 is in the shape of an interdigitated finger, and the third driving valve 134 and the liquid flow pipe 113 between the adjacent culture units 111 form a micro valve at the intersection thereof to realize the separation of the culture unit 111 at the upper end; The linear channel is formed to be wide at the intersection with the liquid flow conduit 114, and not only the closing of the lower end opening of the culture unit 11 but also the separation between the lower ends of the culture unit 111 can be achieved.

Explanation of the parameters of each layer: In the culture layer 11, except that the width of the two branch pipes of the culture unit 111 is 50 μm, the width of the other pipes is 100 μm. The culture unit 111 has a long side of 7000 μm and a short side of 300 μm. In the driving layer 13, the width of the circulating drive pump 131 is 150 micrometers, and the second driving valve 133, the first driving valve 132, and the third driving valve 134 are divided into two parts, the narrow pipe width is 30 micrometers, and the width of the pipe is wide. 100 micrometers, wherein the intersection of the narrow conduit with the conduit in the culture layer 11 does not constitute a microvalve. The fourth drive valve 135 has a width of 100 microns. All pipes are 10 microns deep. The elastic diaphragm layer 12 has a thickness of 20 μm.

The operation principle of the microfluidic solution concentration generating chip 10 is to first inject the solution A into all the culture units 111 until it is full, and then pressurize the fourth driving valve 135 and the first driving valve 132 to close the liquid flow pipe 114 that it traverses. And the culture unit 111, and injecting the solution B into the liquid flow conduit 112, the solution B entering the annular closed culture unit 111 in the fourth drive valve 135 and the first drive valve The pipe between the 132, at the same time, the solution A originally present in the pipe is flushed out, and then the micro-valve controlled by the second drive valve 133 and the third drive valve 134 is closed, so that the same culture unit 111 exists simultaneously. Solution A and Solution B, the two solutions are separated by a microvalve controlled by a first drive valve 132. The volume of the solution B is the volume of the portion of the culture unit 111 closed by the second drive gate 133 and the microvalve controlled by the first drive valve 132. The volume of the solution A is the volume of the entire culture unit 111 minus the volume occupied by the solution B. After the microvalve controlled by the first driving valve 132 is opened, and the micropump composed of the circulating driving pump 131 is started, the solution A and the solution B are circulated and mixed in the annular closed culture unit 111. Since the liquid flow conduit 112 can be disposed at different positions to connect the adjacent two culture units 111, the volume of the second culture valve 111 and the first drive valve 132 controlling the micro-valve closed portion of the culture unit 111 can be varied as required, and thus can be generated A variety of specific concentration gradients. The solution A and the solution B may be a true solution such as a glucose solution, a peptone solution or water, or may be a suspension containing particles such as microorganisms. Solution A and solution B may be different solutions, or may be solutions of the same solution but different concentrations.

Fig. 2 is a plan view showing a microfluidic solution concentration generating chip in a second embodiment of the present invention. As shown in Fig. 2, in the second embodiment of the present invention, the first driving valve 232 is a linear channel, and the angle between the first driving valve 232 and the culture unit 211 is not 90 degrees. The flow conduit 212 connects the culture unit 211, and the flow conduit 212 is disposed in parallel with the first drive gate 232.

Other structures are the same as those in the first embodiment, and will not be described again.

Fig. 3 is a plan view showing a microfluidic solution concentration generating chip in a third embodiment of the present invention. Referring to Fig. 3, in the third embodiment of the present invention, the first driving valve 332 is a stepped channel, and each of the driving valve units intersects with two branching channels of one of the culture units 311 to constitute a microvalve. The liquid flow conduit 312 communicates with the culture unit 311, and the junction of the liquid flow conduit 312 and the culture unit 311 is adjacent to the intersection of the first drive valve 332 and the culture unit 311, so that when the lower culture unit passes the solution B, the solution B can Flow in one direction and drain solution A.

Fig. 4 is a plan view showing a microfluidic solution concentration generating chip in a fourth embodiment of the present invention. As shown in FIG. 4, in the fourth embodiment of the present invention, the culture unit 411 is a linear channel, the first drive valve 432 is a linear duct, and the liquid flow duct 412 is disposed parallel to the first drive valve 432. It is easily conceivable that the first actuating valve 432 can also be arranged in a stepped manner.

In the above-described first to fourth embodiments, the microfluidic solution concentration generating chip is capable of rapidly generating a plurality of solution concentrations and performing microbial culture therein. The chip does not need to be added to the pyramidal branch pipe network. The gradient generating unit has a small footprint and is easy to implement arraying. It does not require precise flow rate control or long-term balance. The injection can be used to obtain specific gradient concentrations on demand, which can meet various gradients. Concentration requirements.

Fig. 5a is a plan view showing a microfluidic culture detecting chip in a fifth embodiment of the present invention; and Fig. 5b is a cross-sectional view taken along line 1D in Fig. 5a.

As shown in FIG. 5a and FIG. 5b, the microfluidic culture detecting chip 10 includes a culture layer 11 composed of at least one of a polymer, a hydrogel, a silicon wafer, a quartz, a glass, and a metal material or A plurality of combinations are formed, and preferably, the culture layer 11 is made of polydithiosiloxane.

The culture layer 11 is provided with a culture detecting unit 111 (one culture detecting unit is shown), and the culture detecting unit 111 includes a culture channel 1111 connected end to end, a detection channel 1112 communicating with the culture channel 1111, and The color liquid injection path 1113 communicates with the detection liquid injection path 1113 to the detection channel 1112.

The elastic layer 12 is laminated on the upper side of the culture layer 11, and the elastic diaphragm layer 12 is formed of an elastic polymer material. Preferably, the elastic diaphragm layer 12 is made of polydimethylsiloxane.

A driving layer 13 is formed above the elastic diaphragm layer 12, and the driving layer 13 is formed of at least a combination of any one or more of a polymer, a hydrogel, a silicon wafer, a quartz, a glass, and a metal material. The driving layer 13 is made of polydimethylsiloxane.

A drive pump 131 is disposed on the drive layer 13, and the circulation drive pump 131 is positioned above the culture channel 1111 and intersects the culture channel 1111.

Both ends of the circulating drive pump 131 are connected to the outside. When high-pressure gas is injected into the circulating drive pump 131, the elastic diaphragm layer 12 under the circulating drive pump 131 is bent downward to block the culture channel 1111 below the elastic diaphragm layer 12, When the high pressure gas is withdrawn, the elastic diaphragm layer 12 is restored and the lower culture channel 1111 is connected, which is a microscopically well known in the art of microfluidics.

The elastic diaphragm layer 12 may be a separate layer or a driving layer 13 or a layer of the culture layer 11. section.

The circulation drive pump 131 is two or three parallel pipes, and by sequentially pressing at a specific timing, the liquid in the lower culture channel 1111 can be squeezed for one-way flow, which is a micropump known in the art of microfluidics.

The cyclic drive structure formed by the circulation drive pump 131 and the culture channel 1111 and the principle thereof are disclosed in the Chinese Patent Nos. CN201110316751.0 and CN201110142095.7, and the present embodiment will not be described again.

The drive layer 13 is further distributed with a detection drive valve 132. The detection drive valve 132 includes two drive valves, a first drive valve 1321 and a second drive valve 1322, respectively. The first drive valve 1321 and the second drive valve 1322 are located at the detection. Above the channel 1112 and intersecting the detection channel 1112, as with the principle of the cyclic drive pump 131, the first drive valve 1321 and the second drive valve 1322 form a valve at the intersection with the detection channel 1112.

A sixth driving valve 133 is further disposed on the driving layer 13, and the sixth driving valve 133 is located at an end opening of the culture detecting unit 111. After the culture liquid is injected into the culture detecting unit 111, a high voltage can be introduced into the sixth driving valve 133. The gas is used to achieve closure of the opening of the culture detecting unit 111. It is easily conceivable that in order to achieve the closing of the opening of the culture detecting unit 111, the closing can also be achieved by other means such as sealing.

The microfluidic culture detection chip 10 is operated by first injecting a culture solution containing microorganisms into the culture detecting unit 111, and the microorganisms circulate in the culture channel 1111 with the liquid flow to carry out suspension growth, and some of the culture liquids remain in the detection. In the channel 1112, under the specified time condition, the pressurized gas in the first driving valve 1321 is removed, and the culture liquid reacts with the color developing liquid in the detecting channel 1112, and the generated optical signal is collected by the external optical detector. Thereby, specific substances in the culture solution such as inorganic phosphorus, glucose, and the like are quantified. After the reaction is completed, the cleaning solution is introduced into the coloring solution injection channel 1113, and the tube is cleaned for the next inspection. By the above method, the culture solution in the culture detecting unit 111 can be detected at different time periods.

Specifically, the action relationship of the microfluidic culture detecting chip 10 is as follows:

(1) First, a culture solution containing microorganisms is introduced into the culture detecting unit 111, and the channel 1111 is to be cultured. After detecting that the channel 1112 is filled with the culture solution, the first driving width 1321 and the sixth driving valve 133 are filled with high-pressure gas.

(2) The high-pressure gas is charged into the circulation drive pump 131 at a specific timing to promote the circulation of the culture liquid, and the microorganism culture is started.

(3) Fill the coloring liquid injection channel 1113 with the cleaning liquid to complete the pipeline cleaning.

(4) After the culture for a period of time, the culture liquid component is detected, the second drive valve 1322 is filled with the high pressure gas, the color development liquid is injected into the color development liquid injection channel 1113, the injection is stopped after the pipe is filled, and the first drive valve 1321 is removed. After the medium and high pressure gas is charged with the high pressure gas for a while, after a certain time, the detection channel 1112 above the first drive valve 1321 is optically detected, and the light intensity signal is collected, thereby performing material quantification.

(5) After the detection is completed, the high-pressure gas in the first driving valve 1321 is removed, and the cleaning liquid is filled into the coloring liquid injection channel 1113 to complete the pipeline cleaning for the next detection. Then, the first drive valve 1321 is filled with high-pressure gas, the high-pressure gas in the second drive valve 1322 is removed, and the microbial suspension culture is continued.

Fig. 6a is a plan view showing a microfluidic culture detecting chip in a sixth embodiment of the present invention; and Fig. 6b is a cross-sectional view taken along line 2D in Fig. 6a.

As shown in Figs. 6a and 6b, in the sixth embodiment of the present invention, two culture detecting units 211 are juxtaposed on the culture layer 21, and it is easily conceivable that the number of the culture detecting units 211 may be more than two.

The coloring liquid injection channel 2113 is set to be Z-shaped, that is, the coloring liquid injection channel 2113 is connected to the detection channel 2112 after two times of bending, and the coloring liquid injection between the adjacent culture detecting units 211 Channels 2113 are in communication.

Also disposed on the drive layer 23 is a third drive valve 234 that intersects the chromogenic solution injection channel 2113 and the detection channel 2112, respectively.

In the prior art, the separation between the conventional culture detection units uses an interdigitated pneumatic microvalve, and the superposition between the drive layer and the culture layer needs to be accurately positioned in two dimensions of the horizontal plane. The ideal positioning of the interdigitated pneumatic microvalve is that the two interdigitated microvalves are located in the longitudinal direction (the direction in which the culture unit extends). The two sides of the horizontal liquid pipe are closely arranged in the longitudinal direction but do not overlap with the liquid channel. The horizontally-pronged pin-shaped pneumatic microvalve is to be crossed with the longitudinal liquid pipe, and the length of the interdigital finger is as short as possible, which reduces the occupied chip area and improves the integration degree. However, there are errors in the alignment of the two layers of the chip in the two dimensions of the horizontal plane during the chip fabrication process. In order to prevent the error caused by the processing from affecting the use of the chip, it is necessary to increase the pitch of the interdigital pneumatic valve and the length of the interdigital finger, which inevitably increases the chip area occupied by the micro valve and reduces the integration degree of the chip. In addition, when the two-layer chip pattern slightly differs due to the difference in substrate shrinkage, even if it is precisely aligned in a certain area, the dimensional deviation between the two layers increases as the distance of the alignment point increases, resulting in The volume of each reaction chamber is inconsistent, affecting the accuracy of detection, and even some units are completely unusable for detection.

The Z-shaped coloring liquid injection channel 21 13 combined with the linear third driving valve 234 can also achieve the function of separating the cells, and the assembly precision of each layer is lower, and the upper and lower laminates only need to be parallel in one dimension. There is no need to precisely align the layers in the two dimensions of the horizontal plane, which reduces the difficulty of chip fabrication and helps to increase the number of chip units.

In some embodiments, the driving layer 23 may further be distributed with an interdigitated fifth driving valve 235, and the fifth driving valve 235 and the color developing liquid injection channel 2 U between the adjacent culture detecting units 21 1 respectively. 3 Form a cross. Specifically, the fifth driving valve 235 includes a lateral extending portion 2351 and a longitudinal extending portion 2352, wherein the lateral extending portion 235 1 extends laterally and vertically intersects with the culture detecting unit 21 1 , and the cross-sectional area of the lateral extending portion 235 1 is relatively small, A "microvalve" is formed at the intersection with the culture detecting unit 21 1; the longitudinal extending portion 2352 extends in the longitudinal direction and communicates with the lateral extending portion 235 1, and the longitudinal extending portion 2352 is formed between the adjacent culture detecting units 21 1 and The coloring liquid injection channel 21 13 forms an intersection to realize a "micro-wide" function. When the high-pressure gas is introduced into the fifth driving valve 235, the separation between the culture detecting units 21 1 and the cross-contamination of the fluid between the cells can be reduced. Probability. The longitudinal extension 2352 also forms an intersection at the inlet and the outlet of the coloring liquid injection passage 21 13 to realize a "microvalve" function, and it is easily conceivable that the inlet and the outlet of the coloring liquid injection passage 21 13 can also pass. Sealing is achieved by means of sealing.

Further, a fourth driving valve 236 is disposed on the driving layer 23, the fourth driving valve 236 is located above the culture channel 21 1 1 and intersects the culture channel 21 1 1 , and the fourth driving valve 236 is located in the cyclic driving. Between the pump 231 and the detection drive valve 232.

The culture detecting unit 211 further includes a cleaning liquid injection path 2114 that communicates with the junction of the culture channel 2111 and the detection channel 2112.

In other embodiments, the cleaning fluid injection channel 2114 can also be in communication with the detection channel 2112 and between the two adjacent drive valves in the detection channel 2112. Specifically, the cleaning liquid injection passage 2114 is located between the first drive valve 2321 and the second drive valve 2322. In order to achieve a better cleaning action, the cleaning liquid injection channel 2114 may also be provided in plurality, for example, between the first driving valve 2321 and the second driving valve 2322 and at the junction of the culture channel 2111 and the detection channel 2112. There is a cleaning liquid injection channel 2114, which is not limited in the present invention.

The cleaning liquid injection passage 2114 between the adjacent culture detecting units 211 is in communication. Further, a sixth driving valve 237 is disposed on the driving layer 23, and the sixth driving valve 237 is formed to intersect with the cleaning liquid injection passage 2114 between the adjacent culture detecting units 211, respectively. The sixth drive valve 237 has the same structure as the fifth drive valve 235, and is used to achieve separation between the culture detecting units 211.

Normally, the cleaning liquid is injected into the coloring liquid injection channel 2113 to inject the coloring liquid into the channel 2113, and the reactant in the detecting channel 2112 is flushed out of the pipeline to complete the cleaning. However, when detecting the channel 2112 When the reactant is still difficult to be rinsed, the fourth driving valve 236 needs to be filled with the high pressure gas, and the cleaning liquid is injected into the cleaning liquid injection channel 2114. The cleaning liquid flows through the detection channel 2112 in one direction to complete the entire detection channel 2112. Cleaning. If the microbial suspension culture is to be continued after the cleaning operation is completed, the sixth driving valve 237 and the first driving valve 2321 are filled with high-pressure gas, the high-pressure gas in the fourth driving valve 236 is removed, and the micro-pump driving culture liquid is turned on. Circulating flow, suspension culture.

A seventh driving valve 238 is further disposed on the driving layer 23, and the seventh driving valve 238 is located at the end outlet of the culture detecting unit 211. After the culture liquid is injected into the culture detecting unit 211, a high voltage can be introduced into the seventh driving valve 238. Gas to achieve closure of the outlet of the culture unit 211. It is easily conceivable that in order to achieve the closing of the outlet of the culture detecting unit 211, the closing can also be achieved by other means such as sealing.

Specifically, the action relationship of the microfluidic culture detecting chip 20 is as follows:

(1) The fifth drive valve 235 and the sixth drive valve 237 are first filled with high-pressure gas. (2) Then, a culture solution containing microorganisms is introduced into each culture detecting unit 21 1 , and after the channel 21 1 1 is cultured, and the detection channel 21 12 is filled with the culture liquid, the driving valve 233 and the first driving valve 2321 are charged. The high-pressure gas is introduced, and the high-pressure gas is charged into the circulation-driven pump 231 at a specific timing to promote the circulation of the culture liquid, and the microorganism culture is started.

(3) The high-pressure gas in the fifth driving valve 235 is removed, and the cleaning liquid is filled into the coloring liquid injection passage 21 13 to complete the pipeline cleaning.

(4) After culturing for a period of time, the composition of the culture solution is detected, and the high-pressure gas is charged into the pneumatic control pipes 236, 2321, and 238, and the coloring liquid is injected into the coloring liquid injection channel 21 13 to stop the injection after the pipe is filled. The three-drive valve 234 is filled with high-pressure gas, and the high-pressure gas in the first drive valve 2321 is removed for a period of time and then charged with high-pressure gas again. After waiting for a certain period of time, the flow conduit between the first drive valve 2321 and the third drive valve 234 is waited for a certain period of time. An optical imaging test is performed, and a light intensity signal is collected, and the substance is quantified accordingly.

(5) After the detection is completed, the high-pressure gas in the first driving valve 2321 and the third driving valve 234 is removed, and the cleaning liquid is filled into the coloring liquid injection passage 21 13 to complete the pipeline cleaning for the next detection. Then, the first driving valve 2321 is filled with high-pressure gas, and the high-pressure gas in the fourth driving valve 236 and the second driving valve 2322 is removed, and the microbial suspension culture is continued.

In the technical solutions of the fifth embodiment to the sixth embodiment described above, microbial suspension culture can be realized on the same chip, and the detection of the specific substance content in the culture solution can be performed in a plurality of culture periods. The chip combines the pipeline unit for microbial suspension culture and culture liquid detection, and detects the glucose, inorganic phosphorus and the like by suspending and culturing the microorganism in the culture channel, and then placing part of the culture solution in the detection channel for color reaction. The concentration of bacteria can simultaneously observe the growth of bacteria and the changes of nutrients in the culture medium, which provides a basis for screening microorganism strains. This feature is not possible with the prior art. The chip unit has a small structure, the width and depth of the liquid line are both micron, and the volume of the culture and analysis solution in each unit is nano-scaled, compared to hundreds of microliters in the multi-channel microfluidic microreaction chip of Nicolas Szita. In terms of the reaction unit, the chip unit is further miniaturized, and the number of integrated units per unit area can be greatly improved, and the culture analysis efficiency is higher, and there is no need to install a micro-stirring paddle or a patch sensor, and only a three-layer structure is required. Superimposed, the production process is simpler. The cost is more 氐.

Fig. 7 is a plan view showing the microfluidic solution concentration generation and culture detecting chip in the seventh embodiment of the present invention; Fig. 8 is a cross-sectional view taken along line 1D in Fig. 7.

As shown in FIG. 7 and FIG. 8, the microfluidic solution concentration generation and culture detecting chip 10 includes a culture layer 11 which is at least composed of a polymer, a hydrogel, a silicon wafer, a quartz, a glass, and a metal material. A combination of any one or more is formed. Preferably, the culture layer 11 is made of polydimethenylsiloxane.

A plurality of culture detecting units 111 are arranged side by side on the culture layer 11, and the culture detecting unit 111 is a unit having the same shape and volume, and the units are arranged in parallel. The culture detecting unit 111 includes a culture channel 1111 connected end to end, a detection channel 1112 communicating with the culture channel 1111, and a coloring liquid injection channel 1113, which communicates with the detection channel 1112.

The coloring liquid injection channel 1113 is set to be Z-shaped, that is, the coloring liquid injection channel 1113 is connected to the detection channel 1112 after being bent twice, and the coloring liquid injection channel 1113 between the adjacent culture detecting units 111. Connected.

The liquid flow pipe 112 is connected between the culture detecting unit 111, and the liquid flow pipe 112 includes a plurality of liquid flow pipe units 1121, and each liquid flow pipe unit 1121 is connected between adjacent culture channels 1111, and the liquid flow pipe unit 1121 Extending in the lateral direction and perpendicular to the culture channel 1111, the plurality of liquid flow pipe units 1121 are arranged in a stepped manner.

The culture detecting unit 111 further includes a cleaning liquid injection path 1114 that communicates with the junction of the culture channel 1111 and the detection channel 1112.

The lower ends of all the culture detecting units in are connected in common to the liquid flow pipe 113, and the liquid flow pipe 113 communicates with the outside. The solution can be simultaneously injected into all of the culture detecting units 111 through the liquid flow path 113.

It is easily conceivable that all the culture detecting units 111 can be independent units, that is, the lower ends of the culture detecting unit 111 are not contiguous, and each has an independent inlet, so that different solutions can be injected into the different culture detecting units 111.

The elastic layer 12 is laminated on the upper side of the culture layer 11, and the elastic diaphragm layer 12 is formed of an elastic polymer material. Preferably, the elastic diaphragm layer 12 is made of polydimethicone. A driving layer 13 is formed above the elastic diaphragm layer 12, and the driving layer 13 is formed of at least a combination of any one or more of a polymer, a hydrogel, a silicon wafer, a quartz, a glass, and a metal material. The driving layer 13 is made of polydidecylsiloxane.

Both ends of the circulating drive pump 131 are connected to the outside. When high pressure gas is injected into the circulating drive pump 131, the elastic diaphragm layer 12 under the circulating drive pump 131 is bent downward to block the culture channel 1111 below the elastic diaphragm layer 12, When the high pressure gas is withdrawn, the elastic diaphragm layer 12 is restored and the lower culture channel 1111 is communicated, which is a microvalve known in the art of microfluidics.

The drive layer 13 is further distributed with a detection drive valve 132. The detection drive valve 132 includes two drive valves, a first drive valve 1321 and a second drive valve 1322, respectively. The first drive valve 1321 and the second drive valve 1322 are located at the detection. Above the channel 1112 and intersecting the detection channel 1112, as with the principle of the cyclic drive pump 131, the first drive valve 1321 and the second drive valve 1322 form a "microvalve" at the intersection with the detection channel 1112.

A sixth driving valve 133 is further disposed on the driving layer 13, and the sixth driving valve 133 is located at the bottom end opening (culture liquid inlet) of the culture detecting unit 111, and is located above the liquid flow pipe 113, and is injected into the culture liquid for culture detection. After the unit 111, a high-pressure gas can be introduced into the sixth drive valve 133 to effect closure of the culture solution inlet of the culture detecting unit 111. It is easily conceivable that in order to achieve the closing of the inlet of the culture detecting unit 111, the closing can also be achieved by other means such as sealing. Also disposed on the driving layer 13 is a third driving valve 134 that intersects the coloring liquid injection channel 1113 and the detection channel 1112, respectively.

In the prior art, the separation between the conventional culture detection units uses an interdigitated pneumatic microvalve, and the superposition between the drive layer and the culture layer needs to be accurately positioned in two dimensions of the horizontal plane. The ideal positioning of the interdigitated pneumatic microvalve is that the two interdigitated microvalves are located on both sides of the transverse liquid pipe in the longitudinal direction (the direction in which the culture unit extends), and are arranged longitudinally but not overlapping the liquid channel. The pneumatic micro-valve should be crossed with the longitudinal liquid pipe, and the length of the interdigital finger should be as short as possible to reduce the occupied chip area and improve the integration. However, there are errors in the alignment of the two layers of the chip in the two dimensions of the horizontal plane during the chip fabrication process. In order to prevent the error caused by the processing from affecting the use of the chip, it is necessary to increase the pitch of the interdigital pneumatic microvalve and the length of the interdigital finger, which inevitably increases the chip area occupied by the micro valve and reduces the integration degree of the chip. In addition, when the two-layer chip pattern slightly differs due to the difference in substrate shrinkage, even if it is precisely aligned in a certain area, the dimensional deviation between the two layers increases as the distance of the alignment point increases, resulting in The volume of each reaction chamber is inconsistent, affecting the accuracy of detection, and even some units are completely unusable for detection.

The Z-shaped coloring liquid injection channel 1113 combined with the linear third driving valve 134 can also achieve the function of separating the cells, and the assembly precision of each layer is lower, and the upper and lower laminates only need to be parallel in one dimension, without Accurate alignment of the layers in two dimensions of the horizontal plane reduces the difficulty of chip fabrication and helps to increase the number of chip units.

In some embodiments, the driving layer 13 may further be distributed with an interdigitated fifth driving valve 135, which respectively intersects the color developing liquid injection channel 1113 between the adjacent culture detecting units 111. . Specifically, the fifth driving valve 135 includes a lateral extending portion 1351 and a longitudinal extending portion 1352, wherein the lateral extending portion 1351 extends laterally and vertically intersects the culture detecting unit 111, and the cross-sectional area of the lateral extending portion 1351 is relatively small, and is not cultivated. The detecting unit 111 forms a "microvalve" at the intersection; the longitudinal extending portion 1352 extends in the longitudinal direction and communicates with the lateral extending portion 1351, and the longitudinal extending portion 1352 is formed between the adjacent culture detecting units 111 and the color developing liquid injection channel 1113 The intersection is formed to realize the function of the valve. When the high-pressure gas is introduced into the fifth driving valve 135, the separation between the culture detecting units 111 can be achieved, and the probability of fluid cross-contamination between the units can be reduced. 1352 also forms an intersection at the inlet and the outlet of the coloring liquid injection passage 1113 to realize the "microvalve" function, and it is easily conceivable that the inlet and the outlet of the coloring liquid injection passage 1113 can also be sealed by caulking or the like. .

Further, a fourth driving valve 136 is disposed on the driving layer 13, and the fourth driving valve 136 is located above the culture channel 1111 and intersects with the culture channel 1111, and the fourth driving valve 136 is located at the circulating drive pump 131 and the detection driving valve. Between 132.

In other embodiments, the cleaning fluid injection channel 1114 can also be coupled to the channel 1112 and positioned between adjacent ones of the sensing channels 1112. Specifically, the cleaning liquid injection passage 1114 is located between the first drive valve 1321 and the second drive valve 1322. In order to achieve a better cleaning action, the cleaning liquid injection channel 1114 may also be provided in plurality, for example, between the first driving valve 1321 and the second driving valve 1322 and at the junction of the culture channel 1111 and the detection channel 1112. There is a cleaning liquid injection channel 1114, which is not limited in the present invention.

The cleaning liquid injection passage 1114 between the adjacent culture detecting units in is in communication. Further, a seventh driving valve 137 is disposed on the driving layer 13, and the seventh driving valve 137 is formed to intersect with the cleaning liquid injection passage 1114 between the adjacent culture detecting units 111, respectively. The seventh driving valve 137 has the same structure as the fifth driving valve 135, and is used to realize the separation between the culture detecting units 111.

Normally, the cleaning liquid is injected into the coloring liquid injection channel 1113 to inject the coloring liquid into the channel 1113, and the reactant in the detecting channel 1112 is flushed out of the tube to complete the cleaning. However, when detecting the channel 1112 When the reactant is still difficult to be rinsed, the fourth driving valve 136 needs to be filled with the high pressure gas, and the cleaning liquid is injected into the cleaning liquid injection channel 1114. The cleaning liquid flows through the detection channel 1112 in one direction to complete the entire detection channel 1112. Cleaning. If the microbial suspension culture is to be continued after the cleaning operation is completed, the seventh driving valve 137 and the first driving valve 1321 are filled with high-pressure gas, the high-pressure gas in the fourth driving valve 136 is removed, and the micro-pump driving culture liquid circulating flow is started. , suspension culture.

An eighth driving valve 138 is further disposed on the driving layer 13. The eighth driving valve 138 is located at the end outlet of the culture detecting unit 111. After the culture liquid is injected into the culture detecting unit 111, a high voltage can be applied to the eighth driving valve 138. The gas is closed to the outlet of the culture detecting unit 111. It is conceivable that in order to achieve the closure of the outlet of the culture detecting unit 111, it is also possible to pass the sealant or the like. The way to achieve closure.

The driving layer 13 is further distributed with a ninth driving valve 139, and the ninth driving valve 139 forms an intersection with the culture channel 1111 at a first position A, which divides the culture channel 1111 into an upper culture unit 1115 and a lower culture unit. 116. The ninth drive valve 139 includes a plurality of drive valve units 1391 each of which is located above two adjacent branch channels of the adjacent culture channel 1111, and the drive valve unit 1391 extends in the lateral direction and the culture channel 1111 is vertical, and multiple drive valve units 1391 are arranged in a stepped manner. The junction of the flow conduit unit 1121 and the culture channel 1111 is as close as possible to the first position A.

The driving layer 13 is further distributed with a tenth driving valve 140, which intersects with the liquid flow pipe unit 1121 between the adjacent culture channels 1111, respectively, and forms a micro valve at the intersection to control the flow. The pipe unit 1121 is turned on or off. The tenth driving valve 140 also intersects with the liquid flow conduit 113 and forms a microvalve at the intersection. When the high pressure gas is introduced into the tenth driving valve 140, the tenth driving valve 140 can open the lower end of the culture detecting unit 111. The separation is performed while achieving the separation between the lower ends of the culture detecting unit 111.

The microfluidic solution concentration occurs and the operation principle of the culture detecting chip 10: first, the solution A is injected into all the culture detecting units 111 until it is full, and then the sixth driving width 133 and the ninth driving valve 139 are pressurized to close the liquid that passes through it. The flow conduit 113 and the culture channel 1111 are injected into the liquid flow conduit 112, and the solution B enters the conduit between the sixth drive width 133 and the ninth drive valve 139 in the annular closed culture channel 1111. The solution A originally present in the portion of the pipe is flushed out, and then the valve controlled by the tenth driving valve 140 is closed, so that the solution A and the solution B are simultaneously present in the same culture detecting unit 111, and the two solutions are The nine-valve valve 139 controls the microvalve separation. The volume of the solution B is the volume of the portion of the culture r measuring unit 111 closed by the microvalve controlled by the tenth driving valve 140 and the ninth driving valve 139. The volume of the solution A is the volume of the entire culture detecting unit 111 minus the volume occupied by the solution B. The first drive valve 1321 is closed by pressurization. After the microvalve controlled by the ninth driving valve 139 is opened, and the pump composed of the circulating driving pump 131 is started, the solution A and the solution B are circulated and mixed in the annular culture channel 1111. Since the liquid flow conduit 112 can be disposed at different positions to connect the adjacent two culture channels 1111, the tenth drive valve 140 and the ninth drive valve 139 control the slightly wide closed portion. The volume of the subculture unit 1 1 1 can be varied as required, so that a variety of specific concentration gradients can be produced. The solution A and the solution B may be a true solution such as a glucose solution, a peptone solution or water, or may be a suspension containing particles such as microorganisms. Solution A and solution B may be different solutions, or may be solutions of the same solution but different concentrations. The organism is circulated in the culture channel 1 1 1 1 with the liquid flow, and suspension growth is carried out, and part of the culture solution is left in the detection channel 1 1 12 . Before the detection, the color developing liquid is injected through the coloring liquid injecting channel 113, the third driving valve 134 is pressurized, then the pressurized gas is introduced into the second driving valve 1322, and the pressurization in the first driving valve 1321 is removed. The gas, the culture solution reacts with the color developing solution in the detection channel 1 1 12, and the generated optical signal is collected by an external optical detector to quantify specific substances in the culture solution such as inorganic phosphorus, glucose, and the like. After the reaction is completed, the cleaning liquid is introduced into the cleaning liquid injection passage 1 1 14 or the coloring liquid injection passage 1 1 13 to clean the piping for the next inspection. By the above method, the culture solution in the culture detecting unit 1 1 1 can be detected at different time periods.

In the eighth embodiment of the present invention, the microfluidic solution concentration generation and the culture detecting chip may be provided with only one culture detecting unit, as shown in Fig. 9.

Referring to Fig. 10, in the ninth embodiment of the present invention, the position of the liquid flow conduit 3 12 in the culture channel is adjusted, and the positions of the ninth drive valve 339 and the tenth drive valve 340 are adjusted, and the isocratic concentration can be obtained. distributed.

Referring to FIG. 11, in the tenth embodiment of the present invention, the position of the liquid flow conduit 412 in the culture channel is adjusted, and the positions of the ninth drive valve 439 and the tenth drive valve 440 are adjusted, and a random concentration distribution can be obtained. .

In the technical solutions of the seventh to tenth embodiments described above, the microfluidic solution concentration generating chip is capable of rapidly generating a plurality of solution concentrations and performing microbial culture therein. The chip does not need to be added to the pyramidal branch pipeline network. The gradient generating unit has a small footprint and is easy to implement arraying. It does not require precise flow rate control or long-term balance. The injection is simple, and a specific gradient concentration can be obtained according to requirements, which can meet various gradient concentrations. demand. The microfluidic culture detection chip can also realize batch microbial suspension culture and detection of specific components of the culture solution. The number of integrated chips is up to hundreds of units, and the processing is simple, no need to install paddles or integrated test patches, it is very suitable for a large number of bacteria Quick screening. Figure 13 is a block diagram showing the structure of a microfluidic chip (a liquid flow pipe) in an eleventh embodiment of the present invention.

As shown in FIG. 13, the microfluidic chip comprises a culture layer formed of at least a combination of any one or more of a polymer, a hydrogel, a silicon wafer, a quartz, a glass, and a metal material, preferably The culture layer is made of polydithiosiloxane.

A liquid flow conduit 21 (one liquid flow conduit is shown) and a liquid injection passage 22 are distributed on the culture layer. The liquid injection passage 22 communicates with the liquid flow conduit 21. The liquid flow conduit 21 can be used for the cultivation of microorganisms and cells. The liquid injection passage 22 is Z-shaped and can be used for injection of a coloring liquid, a cleaning liquid, or the like.

As shown in Fig. 14, the number of the liquid flow pipes 21 can also be set to a plurality, and three are shown in Fig. 14.

The elastic layer is laminated on the upper side of the culture layer, and the elastic diaphragm layer is formed of an elastic polymer material. Preferably, the elastic diaphragm layer is made of polydimethicone.

A driving layer is laminated on the upper surface of the elastic diaphragm layer, and the driving layer is formed of at least a combination of any one or more of a polymer, a hydrogel, a silicon wafer, a quartz, a glass, and a metal material. Preferably, the driving layer Made of polydimethylsiloxane.

A drive valve 23 is disposed on the drive layer, the drive valve 23 is linear, and the drive valve 23 intersects the flow conduit 21 and the liquid injection passage 22, respectively. Both ends of the driving valve 23 are connected to the outside. When high-pressure gas is injected into the driving valve 23, the elastic diaphragm layer under the driving valve 23 is bent downward to block the liquid flow conduit 21 and the liquid injection passage 22 below the elastic diaphragm layer. When the high pressure gas is removed, the elastic diaphragm layer recovers, and the lower liquid flow conduit 21 and the liquid injection passage 22 communicate, which is a well-known micro-fluidity in the field of microfluidics.

The elastic diaphragm layer may be a separate layer or a part of the drive layer or the culture layer. The use of the Z-shaped liquid injection passage 22 in combination with the linear drive channel 23 not only realizes the function of separating the liquid flow conduits 21, but also requires lower assembly precision between the layers, and the upper and lower laminates only need to be parallel in one dimension, without Precisely aligning the layers in two dimensions of the horizontal plane, reducing the chip Difficult to make.

Figure 15 is a block diagram showing the structure of a microfluidic chip in a twelfth embodiment of the present invention.

As shown in Fig. 15, in the twelfth embodiment of the present invention, the liquid injection path 32 is bent into a rectangular wave shape a plurality of times so that the drive valve 33 and the liquid injection path 32 are crossed twice. Other structures are the same as those in the eleventh embodiment, and will not be described again.

Figure 16 is a block diagram showing the structure of a microfluidic chip in a thirteenth embodiment of the present invention.

Referring to Fig. 16, in the thirteenth embodiment of the present invention, the liquid injection path 42 is a diagonal line segment, and the drive valve 43 and the oblique line segment are once intersected. Other structures are the same as those of the eleventh embodiment and will not be described again.

In the technical solutions of the eleventh to thirteenth embodiments, the driving valve is respectively formed to intersect with the liquid flow pipe and the liquid injection passage, thereby not only realizing the function of separating between the liquid flow pipes, but also the accuracy requirement of assembly between the layers. Lower, the upper and lower stacks only need to be parallel in one dimension, without the need to precisely align the stack in two dimensions of the horizontal plane, which reduces the difficulty of chip fabrication and helps to increase the number of liquid flow pipelines. 17a to 17d are views showing the structure of a microfluidic microbial culture chip in the fourteenth embodiment of the present invention.

Referring to Figures 17a through 17d, the microfluidic microbial culture chip consists of three structural layers stacked one above the other. The bottom layer is the culture layer 1 10, and the upper layer is the pneumatic control layer 120, and the elastic gas permeable membrane 130 is separated between the two layers. The culture layer 100 is distributed with the culture unit 100, and each of the culture units 100 includes an annular closed pipe 1101 in the culture layer, and interfaces at both ends are connected to the outside of the chip. A gas supply pipe 1202 and a circulation drive valve 1201 are distributed in the pneumatic control layer 120. Both ends of the gas supply pipe 1202 and the circulation drive valve 1201 are connected to the outside. When a high pressure gas is injected into the circulation drive valve 1201 at a specific timing, the elastic gas permeable membrane 130 under the circulation drive valve 1201 is bent downward in order, and is driven. The liquid in the lower annular closed duct 1 101 flows in a specific direction. The gas supply pipe 1202 is one or a plurality of pipes connected to an external air source.

In the microfluidic microbial culture chip, except for the width of the two branch pipes of the annular closed pipe 1 101 being 50 μm, the remaining pipe widths were 100 μm. Annular closed pipe 1 101 Pass. The gas supply line 1202 and the circulation drive valve 1201 are both 100 microns wide and all pipe depths are 10 microns. The elastic gas permeable membrane has a thickness of 20 microns. Each layer was made of polydithiosiloxane and laminated in this order.

Principle description

The closed culture tube 1101 contains a microbial culture solution which is circulated in a one-way circulation under the action of the circulation drive valve 1201 to realize suspension culture of microorganisms. This principle is disclosed in Patent No. 201 1 103 16751. 0, and will not be described again. The gas supply pipe 1202 is an oxygen-passing pipe which passes over the annular closed pipe 1 101. The gas in the gas supply pipe 1202 will diffuse to the ring through the elastic gas permeable membrane 130 at the intersection with the annular closed pipe 1 101. The culture solution of the pipe 1 101 is closed, and further dispersed in the entire culture solution as the liquid flow is circulated. By changing the area and the number of intersections of the gas supply pipe 1202 and the annular closed pipe 1 102, the speed at which the gas diffuses into the culture liquid can be changed, thereby changing the dissolved oxygen concentration in the culture unit, and the gas supply pipe 1202 can also be changed. The concentration of the gas is introduced to change the dissolved oxygen concentration in the culture unit. The same dissolved oxygen concentration can be achieved if the pipe size, traits, and gas content are exactly the same. The gas in the gas supply pipe 1202 may be oxygen or a gas containing oxygen. The gas pressure in the gas supply pipe 1202 may be the same as the external atmospheric pressure, or may be slightly higher than the external atmospheric pressure, but the gas pressure cannot completely make the elastic gas permeable membrane 130 completely. The liquid flow conduit below the closure may also be a negative pressure, but wherein the partial pressure of oxygen should be higher than the partial pressure of oxygen in the liquid in the annular closed conduit 1 101.

Action relationship description

The microbial-containing culture solution is introduced into the annular closed conduit 1 1 01 in each culture unit 100. After the culture solution is filled, the gas supply pipe 1202 is filled with an oxygen-containing gas, and after being inflated, the gas supply pipe 1202 can be closed. The end interface, which maintains normal pressure, can also be filled with oxygen-containing gas to the pneumatically controlled gas supply pipe 1202 at a lower pressure during the cultivation process. The high-pressure gas is charged into the circulation-driven valve 1201 at a specific timing, the culture liquid is pushed to circulate, and the microbial suspension culture is started.

Fig. 18 is a schematic view showing the structure of a microfluidic microbial culture chip in the fifteenth embodiment of the present invention.

As shown in Fig. 18, the microfluidic microbial culture chip comprises one culture unit, and each culture unit An annular closed conduit 2101 is provided in the culture layer, except that the two branch conduits of the annular closed conduit 2101 have a width of 50 micrometers, and the remaining conduits have a width of 100 micrometers. Both ends of the annular closed duct 2101 are in communication with the outside. A gas supply pipe 2202 and a circulation drive valve 2201 are distributed in the pneumatic control layer of the chip, and the width is 100 micrometers. The gas supply pipe 2202 is a pipe that is bent multiple times, and passes through the annular closed pipe 2101 multiple times, all pipe depths. It is 10 microns. The pneumatic control layer is separated from the culture layer by an elastic gas permeable membrane, and the elastic gas permeable membrane has a thickness of 20 micrometers. Each layer was made of polydimethylsiloxane and laminated in this order.

The fifteenth embodiment has the same principle and working relationship as the fourteenth embodiment, and will not be described again.

Fig. 19 is a view showing the structure of a microfluidic microbial culture chip in the sixteenth embodiment of the present invention.

As shown in Fig. 19, the microfluidic microbial culture chip comprises a culture unit, each culture unit comprising an annular closed conduit 3101 located in the culture layer, except that the two branch conduits of the annular closed conduit 3101 have a width of 50 micrometers. The remaining pipes are all 100 microns wide. Both ends of the annular closed duct 3101 are in communication with the outside. A gas supply pipe 3202 and a circulation drive valve 3201 are distributed in the pneumatic control layer of the chip, and the gas supply pipe 3202 is a pipe with branches, which is traversed several times above the annular closed pipe 3101, and all pipes have a depth of 10 micrometers. The pneumatic control layer is separated from the culture layer by an elastic gas permeable membrane, and the elastic gas permeable membrane has a thickness of 20 μm. Each layer was made of polydithiosiloxane and laminated in this order.

The sixteenth embodiment has the same principle and working relationship as the fourteenth embodiment, and will not be described again.

Fig. 20 is a view showing the structure of a microfluidic microbial culture chip in the seventeenth embodiment of the present invention.

As shown in Fig. 20, the microfluidic microbial culture chip comprises a plurality of culture units, each of which comprises an annular closed conduit 4101 located in the culture layer, except that the width of the two branch conduits of the annular closed conduit 4101 is 50 microns. The remaining pipes are all 100 microns wide. The annular closed duct 410 is connected to the outside at both ends. The gas control layer of the chip is distributed with a gas supply pipe 4202 and a circulation drive valve 4201, each having a width of 100 micrometers, and the gas supply pipe 4202 is a pipe with branches, and there are different times of crossing above the different annular closed pipes 4101, all pipe depths. It is 10 microns. The pneumatic control layer is separated from the culture layer by an elastic gas permeable membrane, and the elastic gas permeable membrane has a thickness of 20 micrometers. Polydimethylation Made of silicone, laminated in sequence. This embodiment is capable of producing different dissolved oxygen concentrations in different culture units.

The seventeenth embodiment has the same principle and working relationship as the fourteenth embodiment, and will not be described again.

Figure 21 is a schematic view showing the structure of a microfluidic microbial culture chip in an eighteenth embodiment of the present invention.

As shown in FIG. 21, the microfluidic microbial culture chip comprises a plurality of culture units, each of which comprises an annular closed conduit 5101 located in the culture layer, except that the two branch conduits of the annular closed conduit 5101 have a width of 50 micrometers. The remaining pipes are all 100 microns wide. Both ends of the annular closed duct 5101 are in communication with the outside. A gas supply pipe 5202 and a circulation drive valve 5201 are distributed in the pneumatic control layer of the chip, and the width is 100 micrometers. The gas supply pipe 5202 is a pipe that is bent multiple times, and passes through different annular closed pipes 5101 with different crossing times. , all pipe depth is 10 microns. The pneumatic control layer is separated from the culture layer by an elastic gas permeable membrane, and the elastic gas permeable membrane has a thickness of 20 microns. Each layer was made of polydimethylsiloxane and laminated in this order. This embodiment is capable of producing different dissolved oxygen concentrations in different culture units.

The eighteenth embodiment has the same principle and working relationship as the fourteenth embodiment, and will not be described again.

Fig. 22 is a view showing the structure of a microfluidic microbial culture chip in the nineteenth embodiment of the present invention.

As shown in Fig. 22, the microfluidic microbial culture chip comprises a plurality of culture units, each of which comprises an annular closed conduit 6101 located in the culture layer, except that the width of the two branches of the annular closed conduit 6101 is 50 microns. The remaining pipes are all 100 microns wide. Both ends of the annular closed duct 6101 are in communication with the outside. A gas supply pipe 6202 and a circulation drive valve 6201 are distributed in the pneumatic control layer of the chip, and the width is 100 micrometers. The gas supply pipe 6202 is a pipe of different width, the width of the pipe is 200 micrometers, and the width of the pipe is 50 micrometers. The width of the pipe section passes over different annular closed pipes, all of which are 10 microns deep. The pneumatic control layer is separated from the culture layer by an elastic gas permeable membrane, and the elastic gas permeable membrane has a thickness of 20 microns. Each layer was made of polydimethylsiloxane and laminated in sequence. This embodiment is capable of producing different dissolved oxygen concentrations in different culture units.

In the fourteenth to nineteenth embodiments described above, the gas concentration generation in the microfluidic chip is dependent on Oxygen is formed by the difference in the amount of oxygen diffused into the solution through a gas permeable membrane at multiple intersections or different cross-sectional areas. The dissolved oxygen concentration is more dependent on the number of intersections and the area of the intersection, avoiding the use of pyramids. The gas-mixed distribution structure saves the chip area and improves the integration of the culture unit in the chip. At the same time, it is not necessary to precisely control the inlet gas flow, only need to be accessed, and the operation is simpler. In the process of chip manufacturing, it is easier to cross the pneumatic control pipe and the liquid flow pipe to make the parallel assembly of the two, which reduces the difficulty in manufacturing the chip. At the same time, the technical solution of the present invention can carry out microbial suspension culture while controlling the dissolved oxygen concentration condition, and the type and application range of the culture are further expanded as compared with the prior art only static culture. Such as carbon dioxide, ammonia, acetic acid, etc., is not limited to oxygen. The gas may be a single type of gas or a mixture of two or more types.

The above is only the preferred embodiment of the invention and is not intended to limit the invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention by using the method content disclosed above, including the recombination between different technical solutions, for example, without departing from the scope of the technical solutions of the present invention. The technical solution for the gas concentration generation disclosed in 17 to 22 can also be applied to the technical solutions disclosed in Figs. 1 to 4, all of which fall within the scope of protection of the claims.

Claims

Rights request

1. A microfluidic solution concentration generating chip, including a culture layer (11) and a drive layer (13) arranged in a stack. A plurality of culture units (111) are distributed side by side on the culture layer, and a plurality of culture units (111) are distributed on the drive layer. There is a first drive valve (132), which is characterized in that: the first drive valve intersects with the culture unit at a first position (A), which divides the culture unit into an upper culture unit (1111) and a lower culture unit. A culture unit (1112), at least part of the culture unit has lower culture units with different volumes.

2. The microfluidic solution concentration generating chip according to claim 1, characterized in that: the culture unit is a circulation channel connected end to end, and a circulation drive valve (131) is provided on the driving layer. The circulation drive The valve intersects with the culture unit and drives liquid circulation in the culture unit.

3. The microfluidic solution concentration generating chip according to claim 1, characterized in that: a liquid flow pipe (112) is connected between the culture units, and the connection between the liquid flow pipe and the culture unit is close to the first position.

4. The microfluidic solution concentration generating chip according to claim 3, characterized in that: the driving layer is also provided with a second driving valve (133), and the second driving valve controls the liquid between adjacent culture units. The opening or closing of flow pipes.

5. The microfluidic solution concentration generating chip according to claim 3, characterized in that: the first drive valve is parallel to the liquid flow pipeline.

6. The microfluidic solution concentration generating chip according to claim 1 or 5, characterized in that: the first drive valve is linear or stepped.

7. A microfluidic solution concentration generating chip, including a culture layer, on which a plurality of culture units are distributed side by side, characterized in that: the microfluidic solution concentration generating chip also includes a driving valve, and the driving valve connects each culture unit to It is divided into an upper culture unit and a lower culture unit, and the conduction and cutoff between the upper culture unit and the lower culture unit is controlled. At least part of the lower culture unit of the culture unit has different volumes.

8. A microfluidic culture detection chip, including a layered culture layer (11) and a driving layer (13), characterized in that: at least one culture detection unit (111) is distributed on the culture layer, and each culture layer The detection unit includes a culture channel (1111) connected end to end and a detection channel (1112) connected to the culture channel. A circulation drive pump (131) and a detection drive valve (132) are distributed on the drive layer. Wherein, the circulation drive pump intersects with the culture channel and drives the culture fluid in the culture channel to circulate; the second drive valve includes at least two drive valves (1321, 1322) and both are connected to The detection channels form an intersection, and the detection channels are controlled to be turned on or off at the intersection.

9. The microfluidic culture detection chip according to claim 8, characterized in that: a chromogenic liquid injection channel (1 1 13) is also connected between the culture detection units, and the detection drive valve is located in the circulation Between the driving width and the chromogenic liquid injection channel.

10. The microfluidic culture detection chip according to claim 9, characterized in that: a third driving valve (234) is also distributed on the driving layer, and the third driving valve is connected to the chromogenic liquid injection channel respectively. It intersects with the detection channel to simultaneously control the conduction of the chromogenic liquid injection channel and the detection channel.

11. The microfluidic culture detection chip according to claim 10, characterized in that: the third drive valve is linear.

12. The microfluidic culture detection chip according to claim 8, characterized in that: a fifth drive valve (235) is also distributed on the drive layer, and the fifth drive valve controls the communication between adjacent culture detection units. The on and off of the chromogenic liquid injection channel.

13. The microfluidic culture detection chip according to claim 8, characterized in that: a fourth drive valve (236) is also distributed on the drive layer, and the fourth drive valve is located above the culture channel and An intersection is formed with the culture channel to control the conduction and cutoff of the culture channel at the intersection, and the fourth drive valve is located between the circulation drive valve and the detection drive valve.

14. The microfluidic culture detection chip according to claim 13, characterized in that: a cleaning liquid injection channel (21 14) is also connected between the culture detection units, and the cleaning liquid injection channel is located in the fourth between the drive valve and the detection channel.

15. The microfluidic culture detection chip according to claim 14, characterized in that: the cleaning solution injection channel is connected to the junction of the culture channel and the detection channel.

16. The microfluidic culture detection chip according to claim 14, characterized in that: a sixth drive valve (237) is also distributed on the drive layer, and the sixth drive valve controls cleaning between adjacent culture detection units. The liquid injection channel is turned on or off.

17. The microfluidic culture detection chip according to claim 8, characterized in that: the drive A ninth drive valve (139) is also distributed on the dynamic layer. The ninth drive valve intersects with the culture channel at a first position (A). This first position divides the culture channel into upper culture units (1 1 15 ) and the lower culture unit ( 1 1 16 ).

18. The microfluidic culture detection chip according to claim 17, characterized in that: at least part of the lower culture units of the culture units have different volumes.

19. The microfluidic culture detection chip according to claim 18, characterized in that: a liquid flow pipe (112) is connected between the culture channels, and the connection between the liquid flow pipe and the culture channel close to the first position.

20. The microfluidic culture detection chip according to claim 19, characterized in that: the driving layer is further provided with a tenth driving valve (140), and the tenth driving valve controls the liquid between adjacent culture channels. The opening or closing of flow pipes.

21. The microfluidic culture detection chip according to claim 18, characterized in that: the ninth drive valve is parallel to the liquid flow pipeline.

11. The microfluidic culture detection chip according to claim 17 or 21, characterized in that the ninth drive valve is linear or stepped.

23. A microfluidic culture detection chip, including a culture layer, with at least one culture detection unit distributed on the culture layer, characterized in that: each culture detection unit includes a culture channel connected end to end and a culture channel connected to the culture layer. The detection channel connected with the culture channel, the microfluidic culture detection chip also includes a circulation drive pump and a detection drive valve, the circulation drive pump drives the liquid flow in the culture channel, the detection drive The valve includes at least two 3-zone valves used to control on or off of the detection channel.

24. A microfluidic chip, including a culture layer and a driving layer arranged in a stack, characterized in that: a liquid flow pipe (21) and a liquid injection channel (22) are distributed on the culture layer, and the liquid injection channel is connected to In the liquid flow pipe, a driving valve (23) is distributed on the driving layer, and the driving valves intersect with the liquid flow pipe and the liquid injection channel to simultaneously control the liquid flow pipe and the liquid injection channel. on and off.

25. The microfluidic chip according to claim 24, characterized in that: the driving valve is i Line style.

26. The microfluidic chip according to claim 24, characterized in that: the driving valve and the liquid injection channel form at least two intersections.

27. A microfluidic microorganism culture chip, characterized by including:

A culture layer (110), at least one culture unit (100) is distributed in the culture layer; a pneumatic control layer (120), a gas supply pipeline (1202) is distributed in the pneumatic control layer, and the gas supply pipeline is connected to The culture units intersect, and the gas pressure introduced into the gas supply pipeline is insufficient to close the culture units at the intersection;

An elastic breathable membrane (130) is formed between the culture layer and the pneumatic control layer. The gas in the gas supply pipe enters the solution in the culture unit through the elastic breathable membrane at the intersection.

28. The microfluidic microorganism culture chip according to claim 27, characterized in that: the elastic breathable film constitutes a side wall of the air supply pipeline.

29. The microfluidic microorganism culture chip according to claim 27, characterized in that: the elastic breathable film constitutes a side wall of the culture unit.

30. The microfluidic microorganism culture chip according to claim 27, characterized in that: the culture layer, the pneumatic control layer and the elastic breathable membrane are all made of breathable materials.

3 1. The microfluidic microorganism culture chip according to claim 30, characterized in that: the breathable material is polydimethylsiloxane.

32. The microfluidic microorganism culture chip according to claim 30, characterized in that: at least part of the side wall of the gas supply pipeline is composed of the pneumatic control layer.

33. The microfluidic microorganism culture chip according to claim 30, characterized in that: at least part of the side wall of the culture unit is composed of the culture layer.

34. The microfluidic microorganism culture chip according to claim 27, characterized in that: the culture unit includes an annular closed pipeline, and a circulation drive valve is also distributed in the pneumatic control layer, and the circulation drive valve is connected with the The culture units form a cross and drive the liquid circulation in the culture units.

35. The microfluidic microorganism culture chip according to claim 27, characterized in that: at least a first culture unit and a second culture unit are distributed in the culture layer, and the gas supply pipeline is connected to the third culture unit. The first culture unit and the second culture unit have different crossing times and/or crossing areas.

36. The microfluidic microorganism culture chip according to claim 27, characterized in that: the gas in the gas supply pipeline is selected from oxygen, carbon dioxide or ammonia.