WO2022206023A1 - 一种能够设置化学浓度梯度的微流控芯片及其制备方法和应用 - Google Patents

一种能够设置化学浓度梯度的微流控芯片及其制备方法和应用 Download PDF

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WO2022206023A1
WO2022206023A1 PCT/CN2021/138001 CN2021138001W WO2022206023A1 WO 2022206023 A1 WO2022206023 A1 WO 2022206023A1 CN 2021138001 W CN2021138001 W CN 2021138001W WO 2022206023 A1 WO2022206023 A1 WO 2022206023A1
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channel
microfluidic chip
concentration gradient
forming part
concentration
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French (fr)
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傅雄飞
苏颖彤
何彩云
白阳
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中国科学院深圳先进技术研究院
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • the invention belongs to the field of microfluidics, and in particular relates to a method for establishing a chemical concentration gradient based on a microfluidic chip.
  • Microfluidic technology refers to a set of technologies for manipulating microvolumes (mL, nL, pL) of liquids in artificially fabricated microsystems, with the basic functions of biological, chemical and other laboratories such as sample preparation, reaction, separation and detection.
  • the basic feature and biggest advantage of the ability to scale down to a few square centimeters of chips is the flexible combination and scale integration of multiple unit technologies on an overall controllable tiny platform.
  • Biochemical molecules such as growth factors, protein signals, chemokines, etc. often form chemical concentration gradients and play important roles in many biological processes.
  • microorganisms often face complex and changeable environments in nature. Therefore, many microorganisms have evolved to have efficient chemotactic systems to recognize the concentration of chemicals in the environment, and move along the concentration gradient to achieve the purpose of seeking advantages and avoiding disadvantages.
  • scientists need to establish concentration gradients of chemicals in vitro to mimic the microenvironment in which the organism lives.
  • the existing microfluidic technology for establishing chemical concentration gradients utilizes laminar flow between liquid flows, and mass transfer occurs between parallel liquid flows through diffusion to form a concentration gradient perpendicular to the liquid flow direction.
  • Common microfluidic chip structures include T-shaped structure and Christmas tree structure.
  • the T-shaped structure is to connect two or more microchannels to one microchannel to form a T-shaped structure.
  • the Christmas tree structure is realized by designing multi-layered and branching microchannels. The streams are repeatedly split and merged, eventually forming a mixed concentration gradient in a main channel.
  • Types of traditional concentration gradient generators include Zigmomd chambers, Boyden chemotaxis chambers, Dunn chambers, biogels, and the like.
  • these traditional methods are difficult to achieve precise and controllable regulation of chemical concentration gradients, and the maintenance time is short.
  • the advantage of microfluidic technology is that it can form a stable, controllable, and quantifiable concentration gradient of chemical substances, and it can also design and process the structure of microchannels according to experimental requirements. cell trajectory.
  • the most common concentration gradient generator based on microfluidic technology is the parallel flow generator.
  • the disadvantage of this technology is that the first is that it needs to continuously provide liquid flow to maintain the stability of the concentration gradient, resulting in the consumption of a large amount of reagents; The flow of liquid will generate mechanical force to damage cells or bacteria; third, in order to achieve precise regulation of concentration, complex structural design is often required. Therefore, the prior art lacks a microfluidic chip with a concentration gradient that is simple in structure, can quickly form a stable concentration gradient, and does not need to consume a lot of expensive reagents.
  • the present invention designs a microfluidic chip with a specific size, and only needs to use a certain concentration of agar gel to control the diffusion rate of the compound to generate a stable concentration gradient and maintain stability for a long time, At the same time, the damage to cells caused by liquid flow shear force is reduced.
  • One aspect of the present invention provides a preparation method of a microfluidic chip capable of setting a chemical concentration gradient, the method comprising the following steps:
  • the channel on the microfluidic chip includes a concentration gradient forming part and a sample injection part provided at both ends of the concentration gradient forming part, and the depth of the channel on the microfluidic chip is less than 80 ⁇ m; the channel on the microfluidic chip passes through the PDMS layer with the concentration gradient forming part and the groove of the sample injection part, and the PDMS layer at the sample injection part is punched to the channel to form the sample injection hole, and then connected with the PDMS layer. Glass slides are bonded to form a sealed microfluidic chip;
  • step 4) injecting the active ingredient and/or dye through the injection hole on one side of the channel, and after standing, a microfluidic chip with a concentration gradient is obtained.
  • the cross section of the channel on the microfluidic chip is rectangular.
  • the depth of the channel on the microfluidic chip is 40-55 ⁇ m.
  • the width of the channel on the microfluidic chip is 0.8-2.5 mm.
  • the length of the channel on the microfluidic chip is 0.8-2.5 cm.
  • the aperture of the injection hole is 50%-100% of the width of the channel on the microfluidic chip, preferably 1.1-1.5 mm.
  • the concentration of the agar gel solution is 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%.
  • the number of channels on the microfluidic chip is more than one.
  • the microfluidic chip is provided with a channel, and the channel includes a concentration gradient forming part and inlets disposed at both ends of the concentration gradient forming part.
  • the depth of the channel on the microfluidic chip is less than 80 ⁇ m; the channel on the microfluidic chip passes through the PDMS layer with the concentration gradient forming part and the groove of the sample introduction part, and the PDMS at the sample introduction part.
  • the layer is punched to the channel to form a sample injection hole, which is then bonded with a glass slide to form a sealed channel including a concentration gradient forming part and a sample injection part; and the channel also contains agar gel, and the agar gel is The concentration of glue is 0.15%-0.3%.
  • the cross section of the channel on the microfluidic chip is rectangular.
  • the depth of the channel on the microfluidic chip is 40-55 ⁇ m.
  • the width of the channel on the microfluidic chip is 0.8-2.5 mm.
  • the length of the channel on the microfluidic chip is 0.8-2.5 cm.
  • the pore size of the injection hole is 50%-100% of the width of the channel on the microfluidic chip.
  • the number of channels on the microfluidic chip is more than one.
  • the concentration gradient forming portion of the channel on the microfluidic chip is a rectangular groove in cross-section, and the sample inlet portion and the sample outlet portion are respectively communicated with the concentration gradient forming portion.
  • Another aspect of the present invention provides an application of the above microfluidic chip, which is a tracking device for microorganisms or cells under a low-power microscope.
  • Another aspect of the present invention provides a tracer device for microorganisms or cells, the device comprising the above-mentioned microfluidic chip of the present invention and an observation or recording device, preferably, the observation or recording device is selected from fluorescent inverted microscopes.
  • the advantages of the present invention lie in that the channel design is simple, and there is no need to continuously provide liquid flow. Different concentration gradients can be generated only by controlling the diffusion rate of chemicals by using agar of different concentrations, which can be maintained stably for 12 hours. Since bacteria or cells are not directly exposed to the flow, damage from shear forces is reduced. It can ensure that the concentration gradient remains stable during the three-dimensional motion tracking of the cells in the microchannel of the chip of the present invention.
  • the microfluidic chip of the invention is simple in design, convenient in manufacture, and only needs a small amount of reagents; and by adjusting the size of the channel, a concentration gradient difference of 5-100% can be realized.
  • FIG. 1 is a schematic diagram of the channel structure in the microfluidic chip of the present invention.
  • 1 is the concentration gradient forming part
  • 2 is the injection part
  • 3 is the injection hole.
  • the advantages of the present invention lie in that the channel design is simple, and there is no need to continuously provide liquid flow. Different concentration gradients can be generated only by controlling the diffusion rate of chemicals by using agar of different concentrations, which can be maintained stably for 12 hours. Since bacteria or cells are not directly exposed to the flow, damage from shear forces is reduced. It can ensure that the concentration gradient remains stable during the three-dimensional motion tracking of the cells in the microchannel of the chip of the present invention.
  • the microfluidic chip of the invention is simple in design, convenient in manufacture, and only needs a small amount of reagents. By adjusting the size of the channel, a concentration gradient difference of 5-100% can be achieved.
  • Fig. 2 is the gray value analysis of the concentration gradient formed by the fluorescent dye in the channel. Using fluorescent dyes to trace the chemical gradient, it can be seen under the microscope that the fluorescence intensity in the channel decreases sequentially from one end of the fluorescent dye injected to the other. The distribution data of fluorescent dyes were analyzed by gray value, and the concentration changes of fluorescent dyes in the channel were obtained. Figure 3 shows the concentration gradient of fluorescent dyes with good performance.
  • FIG. 3 is a photo of the actual microfluidic chip in the present invention, wherein the microfluidic chip is provided with 8 channels.
  • a specific embodiment of the present invention provides a preparation method of a microfluidic chip capable of setting a chemical concentration gradient, the method comprising the following steps:
  • the channel on the microfluidic chip includes a concentration gradient forming part and a sample injection part provided at both ends of the concentration gradient forming part, and the depth of the channel on the microfluidic chip is less than 80 ⁇ m;; the channel on the microfluidic chip passes through the PDMS layer with the concentration gradient forming part and the groove of the sample injection part, and the PDMS layer at the position of the sample injection part is punched into the channel to form the sample injection hole, and then It is bonded to a glass slide to form a channel including a concentration gradient forming part and a sample injection part.
  • step 4) injecting the active ingredient and/or dye through the injection hole on one side of the channel, and after standing, a microfluidic chip with a concentration gradient is obtained.
  • the cross-section of the channel on the microfluidic chip is rectangular.
  • the depth of the channel on the microfluidic chip is 40-55 ⁇ m.
  • the inventors found that the depth of the channel affects the state of the agar gel. When the depth of the channel is too deep, for example, greater than 80 ⁇ m, a stable gel structure cannot be formed, and an effective concentration gradient cannot be formed; When the depth is less than 80 ⁇ m, a stable gel structure can be formed and an effective concentration gradient can be formed.
  • the width of the channel on the microfluidic chip is 0.8-2.5 mm.
  • the length of the channel on the microfluidic chip is 0.8-2.5 cm.
  • the concentration of the agarose gel is very low, if the time for forming the concentration gradient is too long, the effective concentration gradient cannot be formed due to the change of the concentration of the agarose gel, so the length of the channel needs to be controlled to be 0.8-2.5 cm.
  • the pore size of the injection hole is 50%-100% of the width of the channel on the microfluidic chip, preferably 1-1.5 mm.
  • the concentration of the agarose gel solution is 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25% , 0.26%, 0.27%, 0.28%, 0.29%, 0.30%.
  • the concentration of the agar gel solution is very dilute, so with the special channel size, a uniform and stable gel structure can be formed, and then a concentration gradient can be formed after adding dyes or active ingredients.
  • the number of channels on the microfluidic chip is more than one.
  • microorganisms or single cells are added to the same side injection hole or the opposite side injection hole where the active ingredient or dye is added, and the activities of the microorganisms or single cells can be observed.
  • step 1) photolithography is used to obtain a microfluidic chip with channels, which is the same as the conventional process of microfluidic chip fabrication.
  • the A third-party company makes a film that contains structural information.
  • the structure on the film is etched on the silicon wafer, and the SU8-3050 photoresist is used, and the appropriate photolithography parameters are selected according to the properties of the photoresist, and a micro channel with a height of 40-55 ⁇ m is fabricated.
  • the PDMS was flattened on the silicon wafer with the structure, and dried in an oven at 80°C until solidified.
  • the PDMS block is removed from the silicon wafer, the channel structure is cut, and holes are drilled at the position of the injection part to form the injection hole through the upper PDMS to the channel, and then the PDMS structure is formed by plasma bonding. Bonded to clean glass slides.
  • the resulting chips loaded with PDMS structures were used to construct chemical concentration gradients.
  • the heated agar gel solution is injected into the injection hole at one end of the channel, and after it fills the entire chamber, it is left to cool and solidify, and then the fluorescent dye is injected from the other injection port.
  • the size of the chemical gradient and the time required for the formation of the gradient can be controlled by adjusting the concentration of the agarose gel, and the concentration of the agarose gel is set at 0.15%-0.3%.
  • the concentration gradient is established, and then the bacterial sample to be studied is injected into the inlet of the agar gel solution.
  • the chip can be placed under a microscope for real-time observation.
  • the dyes are any dyes having color development under natural light or ultraviolet light, such as fluorescent dyes.
  • the active substances include any substances that have an effect on cells or microorganisms, such as nutrients, drugs, stimulant substances, and the like.
  • the channel on the microfluidic chip includes a concentration gradient forming part and a sample injection part provided at both ends of the concentration gradient forming part, and the depth of the channel on the microfluidic chip is 50 ⁇ m; width is 1.5mm, length is 1 cm; a PDMS layer with a concentration gradient forming part and a groove in the injection part is prepared by photolithography, and the injection hole of the channel is punched at the position of the injection part of the PDMS layer.
  • the diameter of the injection hole is 1.1 mm.
  • the channel on the microfluidic chip includes a concentration gradient forming part and a sample injection part provided at both ends of the concentration gradient forming part, and the depth of the channel on the microfluidic chip is 45 ⁇ m; width is 1.5mm, length is 1cm; a PDMS layer with a concentration gradient forming part and a groove in the injection part is prepared by photolithography, and the injection hole of the channel is punched at the position of the injection part of the PDMS layer.
  • the diameter of the sample hole is 1.1mm, which is then bonded to the glass slide.
  • the channel on the microfluidic chip includes a concentration gradient forming part and a sample injection part provided at both ends of the concentration gradient forming part, and the depth of the channel on the microfluidic chip is 80 ⁇ m, width 1.5mm, length 1 cm; use photolithography to prepare PDMS layer with concentration gradient forming part and groove of injection part, and then punch the injection hole to reach the channel at the position of sample injection part of PDMS layer, The diameter of the injection hole is 1.1mm, and then it is bonded to the glass slide;
  • the channel on the microfluidic chip includes a concentration gradient forming part and a sample injection part provided at both ends of the concentration gradient forming part, and the depth of the channel on the microfluidic chip is 90 ⁇ m, width 1.5mm, length 1 cm; a PDMS layer with a concentration gradient forming part and a groove in the injection part is prepared by photolithography, and then the injection hole of the channel is punched at the position of the injection part of the PDMS layer.
  • the diameter of the injection hole is 1.1mm, and then it is bonded to the glass slide;
  • the channel on the microfluidic chip includes a concentration gradient forming part and a sample injection part provided at both ends of the concentration gradient forming part, and the depth of the channel on the microfluidic chip is 90 ⁇ m, width 2.5mm, length 1cm;
  • the PDMS layer with the concentration gradient forming part and the groove of the injection part was prepared by photolithography, and then the injection hole of the channel was punched at the position of the injection part of the PDMS layer, and the injection hole was The diameter of the sample hole is 1.5mm, and it is bonded to the glass slide;
  • Example 5 The gel state of Example 5 was uniform and stable.
  • a fluorescent dye was added to the injection hole, and the gray value was obtained by analyzing the color of the fluorescent pigment, and the gray value was further analyzed to evaluate whether a concentration gradient was formed.
  • the present invention is used to establish a stable chemical concentration gradient, and the experimental results are as expected, and the gradients with different concentration distributions can be realized by adjusting the concentration of the agar.
  • Figure 2 shows the grey value analysis of the concentration gradient formed by the fluorescent dye in the channel. Using fluorescent dyes to trace the chemical gradient, it can be seen under the microscope that the fluorescence intensity in the channel decreases sequentially from one end of the fluorescent dye injected to the other. The distribution data of fluorescent dyes were analyzed by gray value, and the concentration changes of fluorescent dyes in the channel were obtained.
  • Figure 3 shows the concentration gradient of fluorescent dyes with good performance. And the concentration gradient can be protected for at least 12 hours. And can achieve 5%-100% concentration difference.

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Abstract

一种能够设置化学浓度梯度的微流控芯片及其制备方法和应用，该方法包括：1)获得具有特定尺寸通道的微流控芯片，微流控芯片上的通道包括浓度梯度形成部（1）以及设置在浓度梯度形成部（1）两端的进样部（2），微流控芯片上通道的深度小于80μm；2)配制融化的琼脂凝胶溶液，琼脂凝胶溶液的浓度为0.15％-0.3％；3)将琼脂凝胶溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶。

Description

一种能够设置化学浓度梯度的微流控芯片及其制备方法和应用 技术领域
本发明属于微流控领域，具体涉及一种基于微流控芯片的化学浓度梯度建立的方法。
 背景技术
微流控技术是指在人工制造的微系统内操作微体积（mL、nL、pL）液体的一套技术，具有将生物、化学等实验室的基本功能诸如样品制备、反应、分离和检测等缩微到一个几平方厘米芯片上的能力，其基本特征和最大优势是多种单元技术在整体可控的微小平台上灵活组合、规模集成。
生物化学分子如生长因子、蛋白质信号、趋化物等常形成化学浓度梯度在许多生物过程中发挥重要的作用。尤其是微生物在自然界中往往面临复杂多变的环境，因此许多微生物进化出拥有高效的趋化系统识别环境中的化学物浓度，并沿着浓度梯度运动实现趋利避害的目的。为了研究这些复杂的生物现象，科学家们需要在体外建立化学物质的浓度梯度模拟生物体所处的微环境。现有的用于建立化学浓度梯度的微流控技术是利用液流之间的层流，在并行的液流间通过扩散进行传质，形成垂直于液流方向的浓度梯度。通过控制液体流速和接触时间可快速形成线性的浓度梯度，并在连续提供液流的条件下可长时间保持稳定。常见的微流控芯片结构有T型结构和圣诞树结构，T型结构是将两个或多个微通道连接到一个微通道形成T型结构，圣诞树结构则是设计多层不断分支的微通道实现液流反复分流和汇合，最后在一个主通道中形成混合的浓度梯度。
传统的浓度梯度发生器的种类包括Zigmomd腔、Boyden趋化小室、Dunn腔、生物凝胶等。但这些传统的方法难以对化学浓度梯度实现精确、可控的调节，而且维持的时间较短。与传统的方法相比，微流控技术的优势在于可形成稳定、可控、可量化的化学物质浓度梯度,还可以根据实验需求设计和加工微通道的结构,结合显微镜技术更有利于追踪单细胞的运动轨迹。
目前最常见的基于微流控技术的浓度梯度生成器是并流式生成器，该技术的缺点，第一是需要持续提供液流维持浓度梯度的稳定，造成大量试剂的消耗；第二，连续的液流会产生机械力对细胞或细菌产生伤害；第三，为了实现浓度的精确调控常需要复杂的结构设计。所以现有技术中缺乏结构简单、能快速形成稳定浓度梯度，无需耗费大量昂贵试剂的具有浓度梯度微流控芯片。
 技术问题
本发明为了解决现有技术中的缺陷，设计了特定尺寸的微流控芯片，只需利用一定浓度的琼脂凝胶控制化合物扩散的速率就可产生稳定的浓度梯度，并且保持长时间的稳定，同时减少液流剪切力对细胞带来的损害。
 技术解决方案
本发明一个方面提供了一种能够设置化学浓度梯度的微流控芯片的制备方法，所述方法包括以下步骤：
1）获得具有特定尺寸通道的微流控芯片，所述微流控芯片上的通道包括浓度梯度形成部以及设置中浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度小于80 μm；所述微流控芯片上的通道通过具有浓度梯度形成部以及进样部的凹槽的PDMS层，在进样部位置的PDMS层打孔至通道，形成进样孔，然后与玻片键合，形成密封的微流控芯片；
2）配制琼脂凝胶溶液，所述琼脂溶液的浓度为0.15%-0.3%；
3）将琼脂溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶。
在本发明的技术方案中，还包括步骤4）将活性成分和或染料通过通道的一侧进样孔注入，静置后获得具有浓度梯度的微流控芯片。
在本发明的技术方案中，其中所述微流控芯片上通道的横截面为矩形。
在本发明的技术方案中，所述的微流控芯片上通道的深度为40-55 μm。
在本发明的技术方案中，所述的微流控芯片上通道的宽度为0.8-2.5 mm。
在本发明的技术方案中，所述的微流控芯片上通道的长度为0.8-2.5 cm。
在本发明的技术方案中，所述进样孔的孔径为微流控芯片上通道宽度的50%-100%，优选为1.1-1.5mm。
在本发明的技术方案中，所述琼脂凝胶溶液的浓度为0.15%、0.16%、0.17%、0.18%、0.19%、0.20%、0.21%、0.22%、0.23%、0.24%、0.25%、0.26%、0.27%、0.28%、0.29%、0.30%。
在本发明的技术方案中，微流控芯片上通道的数量为一条以上。
本发明另一个方面提供了另一种能够设置化学浓度梯度的微流控芯片，所述微流控芯片上设置有通道，所述通道包括浓度梯度形成部以及设置在浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度小于80 μm；所述微流控芯片上的通道通过具有浓度梯度形成部以及进样部的凹槽的PDMS层，在进样部位置的PDMS层打孔至通道，形成进样孔，然后与玻片键合，形成密封的、包含浓度梯度形成部以及进样部的通道，；且所述通道内还包含琼脂凝胶，所述琼脂凝胶的浓度为0.15%-0.3%。
在本发明的技术方案中，其中所述微流控芯片上通道的横截面为矩形。
在本发明的技术方案中，所述的微流控芯片上通道的深度为40-55 μm。
在本发明的技术方案中，所述的微流控芯片上通道的宽度为0.8-2.5 mm。
在本发明的技术方案中，所述的微流控芯片上通道的长度为0.8-2.5 cm。
在本发明的技术方案中，所述进样孔的孔径为微流控芯片上通道宽度的50%-100%.。
在本发明的技术方案中，微流控芯片上通道的数量为一条以上。
在本发明的技术方案中，所述的微流控芯片上通道的浓度梯度形成部为横截面呈矩形的长条形凹槽，进样部和出样部分别与浓度梯度形成部联通。
本发明再一个方面提供了一种上述微流控芯片的应用，所述应用为用于低倍镜下微生物或细胞的追踪设备。
本发明再一个方面提供了一种微生物或细胞的示踪设备，所述设备包括本发明上述的微流控芯片以及观察或记录设备，优选地，所述观察或记录设备选自荧光倒置显微镜。
 有益效果
本发明的优点在于通道设计简单，而且不需要持续提供液流，只需利用不同浓度的琼脂控制化学物扩散的速率就可产生不同的浓度梯度，并且稳定保持12小时。由于细菌或细胞不会直接暴露于液流中，减少剪切力带来的损害。能保证细胞在本发明中的芯片微型通道内进行三维运动追踪的过程中浓度梯度保持稳定。本发明的微流控芯片设计简单，制作方便，只需少量的试剂；并通过调节通道的尺寸，可实现5-100%的浓度梯度差。
 附图说明
图1为本发明中微流控芯片中通道结构示意图。其中，1为浓度梯度形成部、2为进样部、3为进样孔。本发明的优点在于通道设计简单，而且不需要持续提供液流，只需利用不同浓度的琼脂控制化学物扩散的速率就可产生不同的浓度梯度，并且稳定保持12小时。由于细菌或细胞不会直接暴露于液流中，减少剪切力带来的损害。能保证细胞在本发明中的芯片微型通道内进行三维运动追踪的过程中浓度梯度保持稳定。本发明的微流控芯片设计简单，制作方便，只需少量的试剂。通过调节通道的尺寸，可实现5-100%的浓度梯度差。
图2为荧光染料在通道中形成的浓度梯度的灰度值分析。利用荧光染料对化学梯度进行示踪展现，显微镜下可以看到通道内的荧光强度从荧光染料注入一端向另一端依次降低。利用灰度值分析荧光染料分布数据，得到荧光染料在通道内的浓度变化，图3显示荧光染料表现良好的浓度梯度。
图3为本发明中微流控芯片的实物照片图，其中该微流控芯片上设置有8条通道。
 本发明的实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
下面结合附图说明本发明的方案。
本发明具体的实施例提供了一种能够设置化学浓度梯度的微流控芯片的制备方法，所述方法包括以下步骤：
1）获得具有特定尺寸通道的微流控芯片，所述微流控芯片上的通道包括浓度梯度形成部以及设置中浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度小于80 μm；；所述微流控芯片上的通道通过具有浓度梯度形成部以及进样部的凹槽的PDMS层，在进样部位置的PDMS层打孔至通道，形成进样孔，然后与玻片键合，形成包含浓度梯度形成部以及进样部的通道，。
2）配制琼脂溶液，所述琼脂溶液的浓度为0.15%-0.3%；
3）将琼脂溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶
在本发明的一些实施例中，还包括步骤4）将活性成分和或染料通过通道的一侧进样孔注入，静置后获得具有浓度梯度的微流控芯片。
在本发明的一些实施例中，其中所述微流控芯片上通道的横截面为矩形。
在本发明的一些实施例中，所述的微流控芯片上通道的深度为40-55 μm。在本发明中，发明人发现通道的深度影响着琼脂凝胶的状态，当通道的深度过深时，例如大于80 μm时，无法形成稳定的凝胶结构，进而无法形成有效的浓度梯度；而深度低于80 μm时，能够形成稳定的凝胶结构，能够形成有效的浓度梯度。
在本发明的一些实施例中，所述的微流控芯片上通道的宽度为0.8-2.5 mm。
在本发明的一些实施例中，所述的微流控芯片上通道的长度为0.8-2.5 cm。在本发明中，由于琼脂凝胶的浓度很低，如果形成浓度梯度的时间过长则导致由于琼脂凝胶浓度变化而无法形成有效的浓度梯度，所以需要控制通道的长度为0.8-2.5 cm。
在本发明的一些实施例中，所述进样孔的孔径为微流控芯片上通道宽度的50%-100%，优选为1-1.5mm。
在本发明的一些实施例中，所述琼脂凝胶溶液的浓度为0.15%、0.16%、0.17%、0.18%、0.19%、0.20%、0.21%、0.22%、0.23%、0.24%、0.25%、0.26%、0.27%、0.28%、0.29%、0.30%。在本发明中，琼脂凝胶溶液的浓度非常稀，这样配合特殊的通道尺寸，可以实现形成均匀的稳定凝胶结构，进而加入染料或活性成分后可以形成浓度梯度。如琼脂浓度再小，则无法形成稳定凝胶结构，而如果浓度太大则导致建立梯度需要的时间过长，时间过长又会导致凝胶结构中水分丧失过多而使得凝胶浓度再次改变。
在本发明的一些实施例中，微流控芯片上通道的数量为一条以上。
在本发明的一些实施例中，在形成凝胶后在加入活性成分或染料的同侧进样孔或异侧进样孔加入微生物或单细胞，并可以观察微生物或单细胞的活动。
在本发明的一些实施例中，步骤1）中，采用光刻技术获得具有通道的微流控芯片，与微流控芯片制作的常规流程相同，将设计好的通道画成CAD文件后，由第三方公司制作成载有结构信息的菲林。通过光刻过程，将菲林上的结构刻蚀在硅片上，利用SU8-3050光刻胶，根据光刻胶性质选择合适的光刻参数，制作成高度为40-55μm的微型通道。最后将PDMS平铺在载有结构的硅片上，置于80℃烘箱烘至凝固。待PDMS凝固后，将PDMS块从硅片上揭取下来，将通道结构切割下来，在进样部位置打孔，形成穿过上层PDMS到达通道的进样孔，然后通过等离子键合将PDMS结构键合于洁净的玻片上。
将得到的载有PDMS结构的芯片用于构建化学物浓度梯度。在通道一端的进样孔注入加热后的琼脂凝胶溶液，待其充满整个腔室后放置冷却凝固，随后将荧光染料从另一进样口注入。此时，可通过调节琼脂凝胶浓度来控制化学梯度的大小及梯度形成所需时间，琼脂凝胶浓度设置在0.15%-0.3%。待一定时间后浓度梯度完成建立，再在注入琼脂凝胶溶液的入口注入需要研究的细菌样品，待细菌在通道内发生趋化运动，就可以将芯片置于显微镜下进行实时观测。
在本发明的一些实施例中，所述的染料为任意具有在自然光或紫外光下显色的染料，例如荧光染料。
在本发明的一些实施例中，所述的活性物质包括任意对细胞或微生物有作用的物质，例如营养物质，药物，刺激性物质等。
实施例1制备微流控芯片
1）获得具有特定尺寸通道的微流控芯片，所述微流控芯片上的通道包括浓度梯度形成部以及设置中浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度50μm；宽度为1.5mm，长度为1 cm；采用光刻方法制备具有浓度梯度形成部以及进样部的凹槽的PDMS层，并在PDMS层进样部的位置打到达通道的进样孔，进样孔的直径为1.1mm。
2）配制融化的琼脂凝胶溶液，所述琼脂溶液的浓度为0.15%；
3）将琼脂溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶。
实施例2 制备微流控芯片
1）获得具有特定尺寸通道的微流控芯片，所述微流控芯片上的通道包括浓度梯度形成部以及设置中浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度45μm；宽度为1.5mm，长度为1cm；采用光刻方法制备具有浓度梯度形成部以及进样部的凹槽的PDMS层，并在PDMS层进样部的位置打到达通道的进样孔，进样孔的直径为1.1mm，然后与玻片键合。
2）配制融化的琼脂凝胶溶液，所述琼脂溶液的浓度为0.15%；
3）将琼脂凝胶溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶。
实施例3 制备微流控芯片
1）获得具有特定尺寸通道的微流控芯片，所述微流控芯片上的通道包括浓度梯度形成部以及设置中浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度80μm，宽度为1.5mm，长度为1 cm；采用光刻方法制备具有浓度梯度形成部以及进样部的凹槽的PDMS层，然后在PDMS层进样部的位置打到达通道的进样孔，进样孔的直径为1.1mm，然后再与玻片键合；
2）配制琼脂凝胶溶液，所述琼脂凝胶溶液的浓度为0.15%；
3）将琼脂凝胶溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶。
实施例4 制备微流控芯片
1）获得具有特定尺寸通道的微流控芯片，所述微流控芯片上的通道包括浓度梯度形成部以及设置中浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度90μm，宽度为1.5mm，长度为1 cm；采用光刻方法制备具有浓度梯度形成部以及进样部的凹槽的PDMS层，然后在PDMS层进样部的位置打到达通道的进样孔，进样孔的直径为1.1mm，然后再与玻片键合；
2）配制琼脂凝胶溶液，所述琼脂凝胶溶液的浓度为0.15%；
3）将琼脂凝胶溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶
对比实施例1-4的琼脂凝胶的状态可知，实施例1-3凝胶均一稳定，而实施例4的凝胶容易破碎，说明深度对于凝胶状态影响较大，需要控制通道的深度在80μm以下。
实施例5制备微流控芯片
1）获得具有特定尺寸通道的微流控芯片，所述微流控芯片上的通道包括浓度梯度形成部以及设置中浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度90μm，宽度为2.5mm，长度为1cm；采用光刻方法制备具有浓度梯度形成部以及进样部的凹槽的PDMS层，然后在PDMS层进样部的位置打到达通道的进样孔，进样孔的直径为1.5mm，在与玻片键合；
2）配制琼脂溶液，所述琼脂溶液的浓度为0.3%；
3）将琼脂溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶。
实施例5的凝胶状态均一稳定。
实施例6应用
采用实施例1制备得到的微流控芯片，在进样孔中加入荧光染料，并通过对荧光颜料颜色的分析获得灰度值，进一步分析灰度值评估浓度梯度是否形成。
本发明用于建立稳定的化学物浓度梯度，实验结果与预期一样，能通过调整琼脂的浓度实现产生不同浓度分布的梯度。图2显示了荧光染料在通道中形成的浓度梯度的灰度值分析。利用荧光染料对化学梯度进行示踪展现，显微镜下可以看到通道内的荧光强度从荧光染料注入一端向另一端依次降低。利用灰度值分析荧光染料分布数据，得到荧光染料在通道内的浓度变化，图3显示荧光染料表现良好的浓度梯度。且该浓度梯度可以保护至少12小时。且能够实现5%-100%的浓度差。

Claims (10)

  1. 一种能够设置化学浓度梯度的微流控芯片的制备方法，其特征在于，所述方法包括以下步骤：
    1）获得具有特定尺寸通道的微流控芯片，所述微流控芯片上的通道包括浓度梯度形成部以及设置中浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度小于80 μm；所述微流控芯片上的通道通过具有浓度梯度形成部以及进样部的凹槽的PDMS层，在进样部位置的PDMS层打孔至通道，形成进样孔；然后与玻片键合，形成包含浓度梯度形成部以及进样部的通道，然后
    2）配制琼脂凝胶溶液，所述琼脂溶液的浓度为0.15%-0.3%；
    3）将琼脂溶液注入微流控芯片的通道中，静置，形成能够形成化学浓度梯度的琼脂凝胶。
  2. 根据权利要求1的制备方法，其特征在于，其还包括，步骤4）将活性成分和或染料通过通道的一侧进样孔注入，静置后获得具有浓度梯度的微流控芯片。
  3. 根据权利要求1的制备方法，其特征在于，其中所述微流控芯片上通道的横截面为矩形。
  4. 根据权利要求1的制备方法，其特征在于，所述的微流控芯片上通道的深度为40-55 μm。
  5. 根据权利要求1的制备方法，其特征在于，所述的微流控芯片上通道的宽度为0.8-2.5 mm。所述的微流控芯片上通道的长度为0.8-2.5 cm。
  6. 一种能够设置化学浓度梯度的微流控芯片，其特征在于，所述微流控芯片上设置有通道，所述通道包括浓度梯度形成部以及设置在浓度梯度形成部两端的进样部，所述微流控芯片上通道的深度小于80 μm；所述微流控芯片上的通道通过具有浓度梯度形成部以及进样部的凹槽的PDMS层，与玻片键合，形成密封的、包含浓度梯度形成部以及进样部的通道，然后在进样部位置的PDMS层打孔至通道，形成进样孔；且所述通道内还包含琼脂凝胶，所述琼脂凝胶的浓度为0.15%-0.3%。
  7. 所述权利要求6的微流控芯片，其特征在于，所述通道的数量为一条以上。
  8. 所述权利要求6的微流控芯片，其特征在于，其通过权利要求1-5任一项的制备方法制备获得。
  9. 一种权利要求6-8任一项所述的微流控芯片的应用，其特征在于，所述应用为用于低倍镜下微生物或细胞的追踪设备。
  10. 一种微生物或细胞的示踪设备，其特征在于，所述设备包括权利要求6-8任一项所述的微流控芯片以及观察或记录设备，优选地，所述观察或记录设备选自荧光倒置显微镜。
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