CN102350379A - Micro fluid control whole blood preprocessing chip based on naturally-deposited filling column - Google Patents
Micro fluid control whole blood preprocessing chip based on naturally-deposited filling column Download PDFInfo
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- 210000004369 blood Anatomy 0.000 title claims abstract description 37
- 239000008280 blood Substances 0.000 title claims abstract description 37
- 239000012530 fluid Substances 0.000 title claims abstract description 8
- 238000007781 pre-processing Methods 0.000 title 1
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 239000011324 bead Substances 0.000 claims abstract description 10
- 239000011325 microbead Substances 0.000 claims abstract description 9
- 239000002002 slurry Substances 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 229920000642 polymer Polymers 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 239000002210 silicon-based material Substances 0.000 claims description 4
- 239000004793 Polystyrene Substances 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 239000000017 hydrogel Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 claims description 2
- 238000007385 chemical modification Methods 0.000 claims description 2
- 238000001312 dry etching Methods 0.000 claims description 2
- 238000000206 photolithography Methods 0.000 claims description 2
- 238000001039 wet etching Methods 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims 1
- 210000000601 blood cell Anatomy 0.000 abstract description 5
- 238000001704 evaporation Methods 0.000 abstract description 2
- 230000008020 evaporation Effects 0.000 abstract description 2
- 238000007873 sieving Methods 0.000 abstract description 2
- 230000017531 blood circulation Effects 0.000 abstract 1
- 238000007689 inspection Methods 0.000 abstract 1
- 238000000605 extraction Methods 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 1
- 238000004159 blood analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000005357 flat glass Substances 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000001259 photo etching Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
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- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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Abstract
本发明提供了一种基于自然沉积的填充柱的微流控全血预处理芯片。该芯片由上下两层组成，上层为具有微结构的盖片，下层为平板基片，上下两层通过封接而形成流体通道或腔室。依靠珠浆中溶液的蒸发，微珠自然沉积在通道中形成填充柱。自然沉积的填充柱作为全血预处理的筛分介质，在全血流经时，血细胞被拦截，而血浆透过填充柱，从而实现简单、快速的全血预处理。毛细管作用力为流体提供驱动力。该芯片组装简单、易于操作、易于便携的特点显示了其在即时检验中应用的前景。
The invention provides a microfluidic whole blood pretreatment chip based on naturally deposited packed columns. The chip is composed of upper and lower layers, the upper layer is a cover sheet with microstructure, the lower layer is a flat substrate, and the upper and lower layers are sealed to form fluid channels or chambers. Relying on the evaporation of the solution in the bead slurry, the microbeads naturally deposit in the channel to form a packed column. The naturally deposited packed column is used as a sieving medium for whole blood pretreatment. When the whole blood flows through, the blood cells are intercepted, and the plasma passes through the packed column, thus realizing simple and rapid whole blood pretreatment. Capillary force provides the driving force for the fluid. The chip is easy to assemble, easy to operate, and easy to carry, which shows the prospect of its application in instant inspection.
Description
技术领域 technical field
本发明涉及在基于自然沉积的填充柱的微流控芯片上进行全血预处理，以毛细管作用力作为流体的驱动力，实现简单、快速地全血中血浆的分离、全血中核酸的提取和特定的血细胞的捕获。The present invention relates to performing whole blood pretreatment on a microfluidic chip based on a naturally deposited packed column, using capillary force as the driving force of the fluid to realize simple and rapid separation of plasma in whole blood and extraction of nucleic acid in whole blood and capture of specific blood cells.
背景技术 Background technique
血液中包含大量的生物信息，可以直接或间接地反映生物体的健康状态和病情变化，对临床有重要的参考意义。血液中大量的干扰物质会影响血液检测的结果，全血预处理是血液分析成功的关键。然而，传统的全血预处理是需要特殊的设备，由专业人士操作，检测的结果需要几小时乃至几天的时间才能获得。Blood contains a large amount of biological information, which can directly or indirectly reflect the health status and disease changes of organisms, and has important reference significance for clinical practice. A large number of interfering substances in blood will affect the results of blood testing, and whole blood pretreatment is the key to the success of blood analysis. However, traditional whole blood pretreatment requires special equipment and is operated by professionals, and it takes hours or even days to obtain the test results.
微流控技术作为一种新型的技术，具有样品用量少、分析时间短、不同操作单元灵活集成的特点。一些研究小组开始探索利用微流控技术实现全血预处理如血浆分离、核酸提取等。Landers研究小组利用围堰的结构或光聚合的方法将核酸提取介质固定于微通道内，进行全血中核酸的提取。Heath研究小组仿照血液在体内毛细管流动的Zweifach-Fung原理构建了微流控血浆分离平台。Ahn研究小组将微珠灌入预先用微坝限定的微通道内制备用于全血分离的填充柱。但是这些方法的局限在于微流控芯片制作复杂，成本高，需要专业人员操作。As a new type of technology, microfluidic technology has the characteristics of less sample consumption, short analysis time, and flexible integration of different operating units. Some research groups have begun to explore the use of microfluidic technology to achieve whole blood pretreatment such as plasma separation and nucleic acid extraction. The Landers research group used the structure of the cofferdam or the method of photopolymerization to fix the nucleic acid extraction medium in the microchannel to extract the nucleic acid from the whole blood. Heath's research group built a microfluidic plasma separation platform based on the Zweifach-Fung principle of capillary flow of blood in the body. Ahn's group prepared packed columns for whole blood separation by filling microbeads into microchannels pre-defined with microdams. However, the limitation of these methods is that the fabrication of microfluidic chips is complicated, the cost is high, and professional operation is required.
本发明设计了一种基于自然沉积的填充柱的微流控全血预处理芯片。它是以自然沉积的填充柱作为全血预处理的筛分介质，毛细管作用力作为流体的驱动力，避免了外接注射泵和复杂的流路连接，实现简单、快速的全血预处理。该芯片组装简单、易于操作、便携的特点显示了其在即时检验中应用的潜力。The present invention designs a microfluidic whole blood pretreatment chip based on naturally deposited packed columns. It uses naturally deposited packed columns as the sieving medium for whole blood pretreatment, and capillary force as the driving force of the fluid. It avoids external syringe pumps and complicated flow path connections, and realizes simple and rapid whole blood pretreatment. The chip is easy to assemble, easy to operate, and portable, showing its potential for instant testing.
发明内容 Contents of the invention
本发明提供了一种基于自然沉积的填充柱的微流控全血预处理芯片，该芯片由上下两层组成，上层为具有微结构的盖片，下层为平板基片，上下两层通过封接而形成流体通道或腔室。自然沉积的填充柱形成于微结构中，填充柱是依靠珠浆中的溶剂的蒸发，微珠自然沉积在通道中。填充柱的微珠材质是二氧化硅、聚苯乙烯、水凝胶。所述自然沉积的填充柱，预先配置的珠浆浓度为50％-300％(wt/vol)，微珠的尺寸为0.5-20μm。填充柱的长度取决于加入珠浆的体积，其长度为1-10mm。The invention provides a microfluidic whole blood pretreatment chip based on naturally deposited filled columns. The chip is composed of upper and lower layers. In turn, fluid channels or chambers are formed. Naturally deposited packed columns are formed in the microstructure. The packed columns rely on the evaporation of the solvent in the bead slurry, and the microbeads are naturally deposited in the channels. The microbead material of packed column is silica, polystyrene, hydrogel. In the naturally deposited packed column, the pre-configured bead slurry concentration is 50%-300% (wt/vol), and the size of the microbeads is 0.5-20 μm. The length of the packed column depends on the volume of bead slurry added, and its length is 1-10mm.
微流控芯片微结构的制作方法为干刻蚀法、湿刻蚀法、光刻蚀法。The fabrication methods of the microstructure of the microfluidic chip include dry etching, wet etching and photolithography.
微流控芯片的材料为玻璃、多聚物、硅材料、金属。The materials of the microfluidic chip are glass, polymer, silicon material and metal.
平板基片材料为玻璃、多聚物、硅材料、金属。The flat substrate material is glass, polymer, silicon material, metal.
毛细管作用力作为流体的驱动力，微流控芯片的盖片或基片需要亲水化处理。Capillary force acts as the driving force of the fluid, and the cover or substrate of the microfluidic chip needs to be hydrophilized.
微流控芯片的盖片或基片亲水化处理的方法为化学修饰、UV/臭氧、氧等离子体。The methods for hydrophilizing the cover or substrate of the microfluidic chip include chemical modification, UV/ozone, and oxygen plasma.
本发明提供的基于自然沉积的填充柱的微流控全血预处理芯片，自然沉积的填充柱能够拦截全血中的血细胞，而血浆可以透过填充柱，从而实现全血中血浆的提取。The present invention provides a microfluidic whole blood pretreatment chip based on a naturally deposited packed column. The naturally deposited packed column can intercept blood cells in the whole blood, and the plasma can pass through the packed column, thereby realizing the extraction of the plasma in the whole blood.
本发明提供的基于自然沉积的填充柱的微流控全血预处理芯片，它不仅可以用于全血中血浆的分离，而且可以用于全血中核酸的提取和特定的血细胞的捕获。The microfluidic whole blood pretreatment chip based on naturally deposited packed columns provided by the present invention can not only be used for the separation of plasma in whole blood, but also can be used for the extraction of nucleic acids in whole blood and the capture of specific blood cells.
附图说明 Description of drawings
图1基于自然沉积的填充柱的微流控全血预处理芯片分析示意图。Figure 1 Schematic diagram of microfluidic whole blood pretreatment chip analysis based on naturally deposited packed columns.
图中：1盖片；2基片、3全血；4自然沉积的填充柱；5分离的血浆。In the figure: 1 cover sheet; 2 substrate, 3 whole blood; 4 naturally deposited packed column; 5 separated plasma.
图2基于自然沉积的填充柱的微流控全血预处理芯片血浆提取结果图。装配有CCD的工具显微镜记录了血浆提取过程。图中标尺长度为500μm。Fig. 2 is a picture of the plasma extraction results of the microfluidic whole blood pretreatment chip based on naturally deposited packed columns. A tool microscope equipped with a CCD recorded the plasma extraction process. The length of the scale bar in the figure is 500 μm.
具体实施方式 Detailed ways
下面的实施例将对本发明予以进一步的说明，但并不因此而限制本发明。The following examples will further illustrate the present invention, but do not limit the present invention thereby.
实施例Example
芯片是由上下两层组成：具有微结构的盖片和基片。盖片上微结构的制作是先利用光蚀刻技术制作SU-8模板，然后将多聚物浇注于模板上聚合成型，最后将印记有微结构的多聚物从模具上剥离。基片是平板玻璃。盖片或基片用氧等离子体处理，使之亲水。最后盖片和基片可逆的封接在一起，形成微流体通道。将不同材质的微珠干粉(二氧化硅、聚苯乙烯、水凝胶)分散于溶剂中，配置成珠浆，其浓度为50％-300％(wt/vol)。然后将充分混匀的珠浆加载于微通道的进样口处，在毛细力的作用下，虹吸进入微流体通道。珠浆的引入量决定了最终制作的填充柱的长度。之后静置使珠浆中的溶剂蒸发。随着溶剂的蒸发，微珠沉积在通道内形成密实的填充柱。10μl的全血滴加于进样口，在毛细力的作用下，血液进入填充柱，其中血细胞被拦截，而血浆穿透填充柱。分离的血浆澄清，呈淡黄色，显示没有溶血发生，并且没有受到微珠的污染。The chip is composed of upper and lower layers: a cover sheet with a microstructure and a substrate. The fabrication of the microstructure on the cover slip is to use photoetching technology to make the SU-8 template, then pour the polymer on the template for polymerization, and finally peel off the polymer with the microstructure printed on it from the mold. The substrate is flat glass. The cover slip or substrate is treated with oxygen plasma to render it hydrophilic. Finally, the cover sheet and the substrate are reversibly sealed together to form a microfluidic channel. Microbead dry powders of different materials (silicon dioxide, polystyrene, hydrogel) are dispersed in a solvent to form a bead slurry with a concentration of 50%-300% (wt/vol). Then the well-mixed bead slurry is loaded on the injection port of the microchannel, and under the action of capillary force, it is siphoned into the microfluidic channel. The amount of bead slurry introduced determines the length of the final packed column. Afterwards, let stand to evaporate the solvent in the bead slurry. As the solvent evaporates, the microbeads deposit within the channel to form a densely packed column. 10μl of whole blood is dripped into the injection port. Under the action of capillary force, the blood enters the packed column, where the blood cells are intercepted, and the plasma penetrates the packed column. The separated plasma was clear and pale yellow, showing no hemolysis and no bead contamination.
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| CN103285949A (en) * | 2013-05-27 | 2013-09-11 | 苏州扬清芯片科技有限公司 | Micro-fluidic serum extracting chip |
| CN106483276A (en) * | 2016-09-29 | 2017-03-08 | 大连理工大学 | A kind of optofluidic blood cell micro imaging system based on smart mobile phone |
| CN106574883A (en) * | 2014-03-05 | 2017-04-19 | 赛拉诺斯股份有限公司 | Method and apparatus for sample collection and sample separation |
| CN106841591A (en) * | 2017-02-27 | 2017-06-13 | 同昕生物技术（北京）有限公司 | The test card that a kind of capillary force drives |
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| CN109012773B (en) * | 2018-08-06 | 2021-01-05 | 西北工业大学 | Preparation method and functionalization method of micro-fluidic chip for photoinduced infiltration |
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Application publication date: 20120215 |