微流体声导术，使用适配子亲和磁珠对革兰氏阴性菌进行流式分离
Summary
本文介绍了使用微流体声传技术以及适配体修饰的微珠的微流控声传导芯片的制造和操作，这些微孔片可用于从培养基中快速，有效地分离革兰氏阴性菌。
Abstract
本文介绍了使用微流体声传技术以及适配体修饰的微珠的微流体声传芯片的制造和操作，这些微珠可用于从培养基中快速，有效地分离革兰氏阴性细菌。这种方法使用长方形微通道的混合物提高了分离效率。在该系统中，样品和缓冲液通过流量控制器注入入口端口。对于磁珠定心和样品分离，交流电源通过带有功率放大器的函数发生器施加到压电换能器上，以在微通道中产生声辐射力。入口和出口处都有一个分叉通道，可以同时进行分离、纯化和浓缩。该装置的回收率为>98%，纯度为97.8%，剂量浓度为10倍。这项研究表明，回收率和纯度高于现有的分离细菌方法，表明该装置可以有效地分离细菌。
Introduction
除了基于介电转移、磁电泳、磁透镜提取、过滤、离心微流体和惯性效应以及表面声波1，2的方法之外，正在开发微流体平台以从医疗和环境样品中分离细菌。使用聚合酶链反应（PCR）继续检测致病菌，但通常费力，复杂且耗时3，4。微流体声传导系统是通过合理的通量和非接触式细胞隔离来解决这个问题的替代方案5，6，7。声光照明是一种利用材料通过声波运动的现象来分离或浓缩磁珠的技术。当声波进入微通道时，根据磁珠的大小、密度等对它们进行分类，细胞可以根据悬浮介质7、8的生化和电学性质进行分离。因此，许多声传声学研究已被积极开展9，10，11，最近，在驻场声波微流体中引入由边界驱动的声流引起的声传运动的3D数值模拟12。
各个领域的研究正在研究如何替换抗体2，3。适配子是一种具有高选择性和特异性的目标材料，许多研究正在进行2，9，10，13。与抗体相比，适配子具有体积小、生物稳定性好、成本低、重现性高等优点，正在诊断和治疗应用中进行研究2，3，14.
在这里，本文描述了一种微流体声传电技术方案，可用于使用适配体修饰的微珠从培养基中快速，有效地分离革兰氏阴性（GN）细菌。该系统通过单压电致动产生二维（2D）声驻波，方法是同时刺激长矩形微通道内的两个正交共振，以将适配体连接的微珠对准并聚焦在节点和反节点点上，以实现分离效率2，11，15，16.入口和出口处都有一个分叉通道，可以同时进行分离、纯化和浓缩。
该方案有助于细菌性传染病的早期诊断，以及通过实时水监测对致病性细菌感染的快速，选择性和敏感反应。
Protocol
1. 微流控声电穿刺芯片设计 注意： 图1 显示了通过声传电从微通道中分离和收集目标微珠的示意图。微流体声传技术芯片采用CAD程序设计。 设计一种微流控声输电芯片，该芯片使用适配体修饰的磁珠和链霉亲和素包覆的聚苯乙烯（PS）磁珠的混合物，对应于细菌的大小，以研究设备的分离性能。 设计一种微流体声传导芯片，…
Representative Results
图5 显示了磁珠流与PZT电压（OFF、0.1 V、0.5 V、5 V）的函数关系。在本研究中引入的声传芯片的情况下，证实随着PZT电压的增加，10μm大小的磁珠的中心浓度增加。大多数10μm大小的磁珠集中在PZT电压的5 V的中心。通过这一结果，在单通道函数发生器中产生了3.66 MHz的谐振频率，并使用功率放大器将一般信号放大了16 dB（约9倍）。 表3 显示了将1…
Discussion
我们开发了一种声波悬浮微流体装置，用于根据其大小和类型以及适配体修饰的微珠，基于连续运行方法，从培养样品中高速捕获和转移GN细菌。与之前报道的 20、21、22、23、24、25、26 相比，长方形微通道可实现更简单的 2D 声传导术设计和…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了韩国政府（科学和ICT部）资助的韩国国家研究基金会（NRF）的资助。（否。NRF-2021R1A2C1011380）
Materials
| 1 µm polystyrene microbeads | Bang Laboratories | PS04001 | Cell size beads |
| 10 µm Streptavidin-coated microbeads | Bang Laboratories | CP01007 | Aptamer affinity beads |
| 4-inch Silicon Wafer/SU-8 mold | 4science | 29-03573-01 | Components of chip |
| Aptamer | Integrated DNA Technologies | GN3-6' | RNA for bacteria conjugation |
| Borosilicate glass | Schott | BOROFLOAT 33 | Components of chip |
| Centrifuge | Daihan | CF-10 | Wasing particles |
| Cyanoacrylate glue | 3M | AD100 | Attach PZT to microchip |
| Escherichia coli DH5α | KCTC | KCTC2571 | Target bacteria |
| Functional generator | GW Instek | AFG-2225 | Generate frequency |
| High-speed camera | Photron | FASTCAM Mini | Observation of separation |
| Hot plate | As one | HI-1000 | Heating plate for curing of liquid PDMS |
| KOVAX-SYRINGE 10 mL Syringe | Koreavaccine | 22G-10ML | Fill the microfluidic acoustophoresis channel with bubble-free demineralized water. |
| Liquid polydimethylsiloxane, PDMS | Dow Corning Inc. | Sylgard 184 | Components of chip |
| LB Broth Miller | BD Difco | 244620 | Cell culture (Luria-Bertani medium) |
| Microscope | Olympus Corp. | IX-81 | Observation of separation |
| PBS buffer | Capricorn scientific | PBS-1A | Wasing bacteria |
| PEEK Tubes | Saint-Gobain Ppl Corp. | AAD04103 | Inject or collect particles |
| Piezoelectric transducer | Fuji Ceramics | C-213 | Generate specific wave in channel |
| Power amplifier | Amplifier Research | 75A250A | Amplify frequency |
| Pressure controller/μflucon | AMED | AMED-μflucon | Control of air pressure/flow controller |
| Tris-HCl buffer | invitrogen | 15567027 | Wasing particles |
| Tube rotator | SeouLin Bioscience | SLRM-3 | Modifiying aptamer and bead |
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Cite This Article
Choi, H. J., Kim, B. W., Lee, S., Jeong, O. C. Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads. J. Vis. Exp. (188), e63300, doi:10.3791/63300 (2022).