细胞分选能够分离出不同种类的细胞并对其进行研究，在生物医学领域具有重要的应用。传统的细胞分选技术包括流式细胞术、磁性激活细胞分选等，这些技术存在复杂的样品处理以及高昂的成本等缺陷。为了克服这些问题，研究人员使用微流控技术进行细胞分选，与传统方法相比，微流控技术具有高通量、自动化、低成本和精准控制等优势。微流控中常用惯性聚焦技术来进行细胞的操纵，但是由于其聚焦位置可控性差、聚焦模式复杂，限制了其在细胞分选中的应用。惯性和粘弹性聚焦技术由于聚焦模式简单、溶液符合生物样本的粘弹特性等优势，受到了研究人员的广泛关注。但是由于微流控芯片制造中软光刻技术的限制，只能通过牛顿和非牛顿流体产生的二维界面进行分选，限制了样本处理的通量。本文基于惯性和粘弹性聚焦技术结合同轴针头产生的三维鞘流界面，使用商业化组件设计组装了一种即插即用的管式微流控平台，创新性的应用于粒子和细胞的三维鞘流聚焦和分选，提升了样本处理的通量。

针对软光刻技术制造的微流控芯片利用牛顿和非牛顿流体产生的二维界面进行细胞分选的低通量问题，本研究提出了三维鞘流聚焦理论并结合管式微流控，组装并实现了一种低廉易得、即插即用、稳定可调的管式微流控平台。使用同轴针头作为产生鞘液和核液三维界面的发生器，结合惯性和粘弹性聚焦技术应用于粒子和细胞的高通量聚焦和分选。研究了微量毛细管中聚氧化乙烯（Polyoxyethylene，PEO）溶液浓度对18 μm粒子聚焦的影响，当PEO浓度为0.1%时，18 μm粒子只在微量毛细管III中实现聚焦，因此选取微量毛细管III作为管式微流控平台的微通道。使用位移平台调节同轴针头和毛细管的三维位置，使管式微流控平台具有三维同轴的可调性。对3D打印支撑模板进行设计和优化，增加了管式微流控平台的稳定性。

针对管式微流控平台中三维鞘流宽度的控制问题，基于同轴共流的泊肃叶流理论及流体稀物质传递物理场仿真模拟，研究了三维鞘流宽度的变化规律并通过实验进行验证。通过同轴共流的泊肃叶流理论，研究了三维鞘流宽度和流量比之间的关系式，为粒子和细胞的三维鞘流聚焦和分选提供了指导。通过流体稀物质传递物理场仿真模拟，分别研究了平台中核液鞘液流量比以及总流量对流体速度场和浓度场的影响，通过浓度场计算了三维鞘流宽度，发现三维鞘流宽度随着流量比发生变化，而和总流量无关。通过实验研究了流量比和三维鞘流宽度的对应关系，当流量比为3和4时，三维鞘流宽度分别为21 μm和18.5 μm，较适合18 μm粒子和12 μm、6 μm粒子的分选。三种方式下得出的三维鞘流宽度展示出了较一致的数值和变化规律，验证了本研究可以通过控制流量比来调控三维鞘流宽度，进而实现粒子基于尺寸的三维鞘流聚焦和分选。

针对管式微流控平台的性能优化和应用，研究了PEO浓度、核液鞘液、流量比以及总流量对粒子三维鞘流聚焦和分选的影响，将优化后的管式微流控平台应用于细胞中，实现了细胞的三维鞘流聚焦和分选。当总流量为700 μL/min，核液鞘液流量比为3，核液通入0.01%的PEO溶液、三维鞘液通入掺有18 μm、12 μm和6 μm混合粒子的甘油溶液时，18 μm粒子能够从三维鞘流中进入到核液并实现单线聚焦，聚焦半峰宽仅为19.9 μm。而12 μm和6 μm粒子被滞留在三维鞘流中且和18 μm粒子的聚焦位置距离较远，分别为53.72 μm和57.23 μm。相较于通过牛顿和非牛顿流体产生的二维界面进行分选的传统微流控芯片，样本处理的通量从7 μL/min提高到175 μL/min，提升了25倍。展示了管式微流控平台进行粒子的高通量三维鞘流聚焦和分选。进行MDA-MB-231和SK-BR-3肿瘤细胞的三维鞘流聚焦实验，实现了两种细胞的单线聚焦。使用18 μm粒子添加到仅稀释20倍的兔全血中进行三维鞘流聚焦实验，结果显示18 μm粒子在通道中心单线聚焦，而血细胞在三维鞘流中环形分布。初步验证了平台三维鞘流聚焦和分选细胞的性能。

本研究基于惯性和粘弹性聚焦技术，结合同轴针头产生的三维鞘流界面，设计组装了一种管式微流控平台，通过控制三维鞘流宽度的大小，可应用于粒子和细胞的高通量三维鞘流聚焦及分选。管式微流控平台使用商业化部件进行组装，价格低廉、操作简便、即插即用，可以应用到肿瘤细胞、细菌及外泌体的分选中，为生物医学中疾病的诊断和治疗提供新的解决方案。

Cell sorting has important applications in the biomedical field as it can isolate different types of cells and study them. Traditional cell sorting techniques, including flow cytometry and magnetically activated cell sorting, have drawbacks such as complex sample handling and high costs. To overcome these problems, researchers use microfluidics for cell sorting, which has the advantages of high throughput, automation, low cost and precise control compared to traditional methods. Inertial focusing techniques are commonly used in microfluidics for cell manipulation, but their poor controllability of focusing position and complex focusing patterns limit their application in cell sorting. Inertial and viscoelastic focusing techniques have received a lot of attention from researchers due to the advantages of simple focusing patterns and solutions that match the viscoelastic properties of biological samples. However, due to the limitation of soft lithography in microfluidic chip fabrication, the binning can only be performed through the two-dimensional interface generated by Newtonian and non-Newtonian fluids, which limits the throughput of sample processing. In this paper, we design and assemble a plug-and-play tubular microfluidic platform based on inertial and viscoelastic focusing techniques combined with a 3D sheath flow interface generated by a coaxial needle using commercially available components, and innovatively apply it to 3D sheath flow focusing and sorting of particles and cells to enhance the throughput of sample processing.

 

To address the low throughput problem of cell sorting by microfluidic chips fabricated by soft lithography using two-dimensional interfaces generated by Newtonian and non-Newtonian fluids, this study proposes the theory of three-dimensional sheath flow focusing and combines it with tubular microfluidics to assemble and realize a cheap, easy-to-use, plug-and-play, stable and adjustable tubular microfluidic platform. A coaxial needle was used as a generator to generate the three-dimensional interface between the sheath and nuclear fluids, and combined with inertial and viscoelastic focusing techniques to apply to high-throughput focusing and sorting of particles and cells. The effect of the concentration of polyoxyethylene oxide (PEO) solution in the microcapillary on the focusing of 18 μm particles was investigated. At a PEO concentration of 0.1%, 18 μm particles were focused only in microcapillary III, which was therefore selected as the microchannel for the tubular microfluidic platform. The three-dimensional position of the coaxial needle and capillary was adjusted using a displacement stage to make the tubular microfluidic stage with three-dimensional coaxial tunability. The design and optimization of the 3D printed support template increases the stability of the tubular microfluidic platform.

 

The 3D sheath flow width control problem in the tubular microfluidic platform is investigated and verified experimentally based on the Poisson lobe flow theory of coaxial co-flow and the simulation of the physical field of fluid rarefaction transfer. The relationship between the three-dimensional sheath flow width and the flow ratio is studied by the Poisson lobe flow theory of coaxial co-flow, which provides guidance for the three-dimensional sheath flow focusing and sorting of particles and cells. By simulating the physical field of fluid dilute matter transfer, the effects of the flow ratio of nuclear liquid sheath fluid and the total flow rate on the fluid velocity field and concentration field in the platform were investigated respectively. 3D sheath flow width was calculated by the concentration field, and it was found that the 3D sheath flow width changed with the flow ratio, but was independent of the total flow rate. The correspondence between the flow ratio and the 3D sheath width was investigated experimentally, and the 3D sheath widths were 21 μm and 18.5 μm when the flow ratios were 3 and 4, respectively, which were more suitable for the sorting of 18 μm particles and 12 μm and 6 μm particles. The three-dimensional sheath flow widths obtained in the three ways show consistent values and variation patterns, which validate that the three-dimensional sheath flow widths can be regulated by controlling the flow ratios to achieve size-based three-dimensional sheath flow focusing and sorting of particles in this study.

 

For the performance optimization and application of the tubular microfluidic platform, the effects of PEO concentration, nuclear fluid sheath, flow rate ratio and total flow rate on particle 3D sheath flow focusing and sorting were investigated, and the optimized tubular microfluidic platform was applied to cells to achieve 3D sheath flow focusing and sorting of cells. When the total flow rate was 700 μL/min, the flow ratio of nuclear fluid to sheath fluid was 3, and the nuclear fluid was fed with 0.01% PEO solution and the 3D sheath fluid was fed with glycerol solution mixed with 18 μm, 12 μm and 6 μm particles, the 18 μm particles could enter the nuclear fluid from the 3D sheath flow and achieve single-line focusing with a focusing half-peak width of 19.9 μm. The 12 μm and 6 μm particles were trapped in the 3D sheath flow and were separated from the 18 μm particles. The sample throughput was increased from 7 μL/min to 175 μL/min, a 25-fold improvement compared to conventional microfluidic chips that sort through the two-dimensional interface between Newtonian and non-Newtonian fluids. A tubular microfluidic platform for high-throughput three-dimensional sheath flow focusing and sorting of particles is demonstrated. Performed 3D sheath flow focusing experiments on MDA-MB-231 and SK-BR-3 tumor cells, and achieved single-line focusing of both cells. 3D sheath flow focusing experiments using 18 μm particles added to rabbit whole blood at only 20-fold dilution showed that the 18 μm particles were focused in a single line in the center of the channel, while the blood cells were distributed in a circular pattern in the 3D sheath flow, which initially validated the performance of the platform for 3D sheath flow focusing and cell sorting.

 

In this study, a tubular microfluidic platform was designed and assembled based on inertial and viscoelastic focusing techniques, combined with the 3D sheath flow interface generated by a coaxial needle, which can be applied to high-throughput 3D sheath flow focusing and sorting of particles and cells by controlling the size of the 3D sheath flow width. The tubular microfluidic platform is assembled using commercially available components and is inexpensive, easy to operate, plug-and-play, and can be applied to the sorting of tumor cells, bacteria, and exosomes, providing a new solution for the diagnosis and treatment of diseases in biomedicine.

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