样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示
Summary
航天血液诊断需要创新。少数示威已经公布出在飞行中，减少了重力医疗诊断技术。这里我们提出了结构和操作一个抛物线飞行试验台上对护理点的一个原型流式细胞仪的设计，与组分和制备策略适应其它设置方法。
Abstract
直到最近，收集宇航员的血液样本中飞行，运到陆地上的航天飞机，并在地面的实验室进行分析。如果人类前往超越低地球轨道，走向太空就绪，点的护理的过渡（POC）测试是必需的。这样的测试必须是全面的，容易降低重力环境下进行，并且不会受到发射和航天的压力。无数POC设备已经被开发，以模仿实验室规模的对应，但最有窄的应用和一些具有在飞行中，减少的重力环境可证实的用途。事实上，在降低重心的生物医学诊断的示威完全限制，使得组件的选择和一些后勤方面的挑战很难寻求测试新技术的时候进场。为了帮助填补这一空白，我们提出了模块化方法的原型血液诊断设备的建设和运营及其相关的P抛物飞行试验台符合标准的飞行试验机载抛物线飞行，减少了重力飞机。该方法首先侧重于钻机装配在飞机上，减少了重力流式细胞仪的测试和同伴微流控芯片的混合。组件是适合于其他的设计和一些定制组件，比如一个微量样品装载器和所述微混合器可以是特别感兴趣的。然后该方法将焦点转移到飞行准备，通过提供指导和建议，以一个成功的飞行试验方面用户培训，标准作业程序（SOP）的发展等问题做准备。最后，在飞行中特定于我们的示范实验步骤进行了描述。
Introduction
目前的空间准备健康诊断的不足之处提出了一个限制因素，以更深入的载人太空探索。诊断需要是全面的，易于在低重力来使用，并且相对地不受发射和航天（ 例如 ，高g力，振动，辐射，温度变化，和机舱压力的变化）的压力。在护理点的检测（POCT）的发展可以通过使用更小的患者的标本（ 例如 ，一个手指刺），更简单和更小的流体学（ 即 ，微流体）转化为有效的航天溶液，并降低电功率的要求，除其他优势。 流式细胞术是有吸引力的方法为在空间的POC，因为该技术的广泛的用途，包括对细胞计数和生物标志物的定量分析，以及显著小型化的潜力。上一页空间相关的流式细胞仪，包括“核包装efficiency'（NPE）的工具，用于同时进行电弧灯诱导荧光和电子音量（库尔特体积）测量1-4中 ，一个相对较小的台式流式细胞仪代表“第一代流式细胞仪中的零重力'5数据的实时流，一个“无鞘微仪”能够4-和5-部分白血细胞（WBC）分类计数用预处理5微升全血样品6-9，以及“光纤为基础的”流式细胞仪最近在国际板载测试空间站10。
通常进行板载了使用近似抛物线飞行弹道模拟失重（ 例如 ，零重力，火星的重力）11的选择水平降低重力飞机的潜在空间应用评估诊断技术。评估是具有挑战性的，因为飞行的机会是有限的，repet微重力可持续的竞争短窗可以使它很难评估方法或通常需要不间断的时间超过20-40秒长的过程，并演示可能需要飞行12-15不要轻易使用额外的设备。此外，在使用或设计，减少了重力作用的体外诊断（IVD）技术先前的示威是有限的，大量的工作仍然未公布。除了 上述的流式细胞仪，在文献中描述的其他空间相关的IVD-技术包括全血染色装置，用于免疫的应用程序16，一个自动化的基于照相机的流式细胞仪12，手持式的临床分析仪，用于集成电势，电流分析法，和电导12,17，一个微流体“T形感应器”装置，用于分析物的定量分析，依赖于扩散为基础的混合和分离18，和一个旋转的'实验室上的CD“诊断平台19,20。初来乍到低重力测试也可能看起来不相关的体外诊断抛物线飞行表演时，试图使设备评估可能的（或搞清楚什么是可能的）。从具有证据充分的飞行准备，飞行中的战略，以及飞行测试设备等以前的医学或生物实验演示包括在表1中 15，21-35。这些可以是信息性由于包含的手册中飞行任务，使用专门的设备，并且实验遏制。
| 类别 | 示例 |
| 紧急医疗 | 气管插管（喉镜引导下，对马尼克在）21，心脏生命支持（麻醉猪）22 |
| 外科护理 | 腹腔镜手术（视频模拟23，对麻醉的猪24,25） |
| 医学影像学或生理学评估 | 超声与下体负压室26，多普勒流量计（头戴式）27，中心静脉压监测器28 |
| 专业的生物设备 | 酶标仪（和飞行中的手套箱中）29，对细胞周期的实验30，显微镜温度控制系统（明场，相位相反，和多通道荧光能力）15，毛细管电泳设备连接到视频显微镜31 |
| 其他 | 植物收割用钳子32，只包含33,34和35鱼类观察 |
表1.抛物线飞行有很好的描述方法/试验示范的例子
为了扩大在前面的例子中，提供更深入地了解成功的飞行演示中，我们提出了模块化和适应性强的程序结构的原型与相关的微混技术的流式细胞仪为抛物线飞行试验台的组成部分和操作。该钻机能装样，微混合和荧光粒子探测的演示，并进行了测试机上，2010年美国航空航天局方便获取的空间环境（FAST）抛物线fligHTS，飞行距离9月29日至十月一日，2010年这些示威活动从一开始，中间和结束拉，分别在指尖大小的血样被载入，稀释或者与试剂混合的潜在设备的工作流程，并通过光学分析检测。流式细胞仪缩放流入一个紧凑的单元需要创新和精心挑选的一部分。定制和现成的，现成的组件是用在这里，所选择的最后一个组件的选择最好的早期近似的，以及可能适用于其他创新的设计。下面的原型组件选择一个大纲，设置是在作为骨架钻机装配支撑结构描述。原型部件被分配的地点，固定，并伴有必要的成功实验的附加组件。注意然后转移到涉及标准操作程序（SOP）的开发，培训，物流等较为抽象的过程。最后，演示，具体程序描述。此处所描述的策略和支撑钻机部件（ 例如 ，显微镜，丙烯酸箱等 ）的选择，尽管执行此处为特定原型，说话相关测试任何血液诊断设备中减小的重力环境的一般问题和挑战。
在航班2010年2月球重力（达到约1/6地球重力）和两个微重力航班原定跨越4天，虽然最终这些被跨3天改期。示威板载了修改私人经营，窄体喷气客机36进行。每个航班提供30-40抛物线，每产生约20秒的引力（大致1.8克），随后由20-25秒的减少重力条件下。被处决的一半抛物线后，飞机停了一段平飞约5-10分钟，以使飞机调头后脑勺朝着陆点，而PErforming的抛物线的其余部分。
Protocol
在这个协议中使用的人类血液样本，采用微创的协议（见致谢）收集与IRB的批准。 1.钻机组装组装样机部件（流体学，光学，控制/数据获取电子设备），用于一个简单的流式细胞仪系统中减少重力条件下使用准备一个加压系统以最小的重量和功率需求驱动系统射流连接一个微型空气泵到一个压差传感器。 为了保持恒定的驱动压力下，使用脉冲宽度?…
Representative Results
对于微混合器示范代表性的结果显示在图7中 ，作为观看由安装在立体显微镜CCD照相机。混合可以在视觉上评价在沿着螺旋的任何点，以及在涉及两组流体实验的出口通道：血液/盐水和蓝色/黄色染料。该二维图像的定量分析可以包括确定在整个信道宽度遮阳均匀性在不同的区域，如图中的其他出版物38-40。见补充图1进一步的细节。见补充图2演示泡处…
Discussion
这里所描述的方法，使主要技术组件（装样，微混合和光学检测）的有效证明在2010年快速抛物线飞行，用比较的结果，以地面试验。这里所描述的培训和SOP方法特别有效，并帮助照亮依靠实践示威不会板载了抛物线飞行工具等“拐杖”的存在。
需要改进的地方，包括遏制和布局。定制亚克力成分可能不是遏制的目的足够强大。在“手套”框被重力过渡期间遭飞行的乘客，并?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
硬件的发展是由美国航空航天局SBIR合同NNX09CA44C和NNX10CA97C支持。对于光学组件和样本装载机演示数据分析是由美国航空航天局的第三阶段合同NNC11CA04C支持。美国航空航天局使用协议IRB＃SA-10-008进行人体血液采集。通过美国国家仪器医疗器械助学计划提供的控制/数据采集软件。模具的微芯片在约翰斯·霍普金斯大学微细加工设备和哈佛大学纳米系统中心作了发言。奥托J.盐水灌装机和卢克贾菲（DNA医学研究所）在2010年夏季美国航空航天局飞行视频的工作人员在飞行过程中每周提供录像辅助机架装配。卡洛斯·巴里恩托斯（DNA医学研究所）提供的照片和图形的援助。特别感谢协助获得空间环境对技术2010计划，美国航天局的低重力办公室，人力适应与对策部，国家航空航天局格伦研究中心，ZIN技术，和人类的研究计划。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Micro air pump | Smart Products, Inc. | AP-2P02A | Max pressure = 6.76 psi; 1.301” x 0.394” x 0.650” , 0.28 oz (8 g); available direct from Smart Products |
| Differential pressure sensor | Honeywell International, Inc. | ASDX015D44R | Range of 0-15psi; 0.974" x 0.550" x 0.440", 0.09 oz (2.565 g); suppliers include Digi-Key and Mouser Electronics |
| Rigid plastic vial (small size) | Loritz & Associates, Inc. | 55-05 | Polystyrene; ID 0.81" (20.6 mm), IH 2.06" (52.4 mm); available direct from LA Container Inc.; similar product available from Dynalab Corp. |
| Rigid plastic vial (larger size) | Loritz & Associates, Inc. | 55-140 | Polystyrene; ID 1.88" (47.6 mm), IH 3.31" (84.1 mm); available direct from LA Container Inc.; similar product available from Dynalab Corp. |
| latex examination gloves | dynarex corporation | 2337 | Middle finger used for latex diaphragm in fluid source vial. Other brands (e.g., Aurelia ® Vibrant ™) acceptable. |
| Optical glue | Norland Products | NOA 88 | Low outgassing adhesive; available direct from Norland; Also available from Edmund Optics Inc. |
| 3-way solenoid valves | The LEE Company | LHDA0531115H | Gas valves, but can function with liquid; 1.29 " L, 0.28 " D. Discontinued product. Similar products available from The LEE Company. |
| Volumetric water flowmeter | OMEGA Engineering inc. | FLR-1602A | Non-contacting flow rate meter strongly preferred. We recommend SENSIRION LG16 OEM Liquid Flow Sensor for flow rates from nl/min up to 5 ml/min. |
| PCD-mini photon detector | Sensl | PCDMini-00100 | For fluorescence detection; available direct from Sensl |
| Accelerometer | Crossbow Technology, Inc. | CXL02LF3 | 3-demensional force detection. Supplied to DMI by NASA. Similar product available from Vernier Software & Technology, LLC. |
| Stereomicroscope | AmScope | SE305R-AZ-E | |
| CCD Camera | Thorlabs | DCU223C | 1024 x 768 Resolution, Color, USB 2.0; available direct from Thorlabs |
| USB and Trigger Cable (In/Out) for CCD Camera | Thorlabs | CAB-DCU-T1 | Available direct from Thorlabs |
| Microbore tubing | Saint-Gobain Corporation | AAD04103 | Tygon®; ID 0.02", OD 0.06", 500ft, 0.02" wall. Suppliers: VWR, Thermo Fisher Scientific Inc. |
| Hollow steel pins | New England Small Tube | (Custom) | 0.025" OD, 0.017" ID, 0.500” L, stainless steel tube, type 304, cut, deburred, passivated; enable microbore tubing connections, chip tubing connections |
| Slide clamp | World Precision Instruments, Inc. | 14042 | Available direct from World Precision Instruments |
| Leur adaptor pieces | World Precision Instruments, Inc. | 14011 | Available direct from World Precision Instruments |
| Silicon wafer | Addison Engineering, Inc. | 6" diameter; for SU-8 mold fabrication | |
| Polydimethylsiloxane (PDMS) elastomer base | Dow Corning | 3097366-1004 | Supplier: Global Industrial SLP, LLC |
| Polydimethylsiloxane (PDMS) elastomer curing agent | Dow Corning | 3097358-1004 | Supplier: Global Industrial SLP, LLC |
| Needle (23 gauge), bevel tip | Terumo Medical Corporation | NN-2338R | Ultra thin wall; 23G x 1.5"; 22G also usable; suppliers: Careforde, Inc., Port City Medical |
| Dispensing needle (23 gauge), blunt tip | CML Supply | 901-23-100 | 23Gx 1"; available from CML Supply |
| Rotary tool | Robert Bosch Tool Corporation | 1100-01 | Dremel® 1100-01 Stylus™ |
| Cover glass | Thermo Fisher Scientific, Inc. | 12-518-105E | Gold Seal™ noncorrosive borosilicate glass; for PDMS chip cover; 24×60 mm; available from Thermo Fisher Scientific, Inc. |
| Vacuum pump | Mountain | MTN8407 | For degassing PDMS; supplier: Ryder System, Inc. |
| Vacuum chamber | Thermo Fisher Scientific, Inc. | 5311-0250 | Nalgene™ Transparent Polycarbonate; available from Thermo Fisher Scientific, Inc. |
| Plasma cleaner | Harrick Plasma | PDC-32G | |
| Hand magnifier | Mitutoyo | 183-131 | Use in reverse direction to enable viewing at ~15". |
| Ethanol | CAROLINA | 861283 | For chip cleaning. Dilute to 70% using millipore water. |
| Water purification system | Thermo Fisher Scientific, Inc. | D11901 | Available direct from Thermo Fisher Scientific, Inc. |
| Optomechanical translation mounts | Thorlabs | K6X | 6-Axis Kinematic Optic Mount; discontinued product; new product (K6XS) available direct from Thorlabs |
| Laptop | Hewlett-Packard | VP209AV | HP Pavilion Laptop running Windows 7 |
| Laptop tray (spring loaded) | National Products, INC. | RAM-234-3 | RAM Tough-Tray™. Can accommodate 10 to 16 inch wide laptops. |
| USB splitter | Connectland Technology Limited | 3401167 | |
| USB Data Acquisition Cards (8 analog input, 12 digital I/O) | National Instruments | NI USB-6008 | 12-Bit, 10 kS/s Low-Cost Multifunction DAQ |
| USB Data Acquisition Cards (16 analog input, 32 digital I/O) | National Instruments | NI USB-6216 | 16-Bit, 400 kS/s Isolated M Series MIO DAQ, Bus-Powered |
| Control/acquisition Software | National Instruments | LabVIEW 2009 | Custom coded National Instruments (NI) LabVIEW |
| 3D Solid Modeling Software | Dassault Systèmes SolidWorks Corp. | SolidWorks 2011 | |
| 2D Modeling Software | AUTODESK | AutoCAD LT 2008 | |
| Vertical equipment rack | (NASA provided) | N/A | |
| Solid aluminum optical breadboard | Thorlabs | MB2424 | 24" x 24" x 1/2", 1/4"-20 Taps; available direct from Thorlabs |
| Industrial grade steel and hardener | The J-B Weld Company | J-B Weld Steel Reinforced Epoxy Glue | |
| Micro-hematocrit capillary | Fisher Scientific | 22-362-574 | inner diamter 1.1 to 1.2 mm |
| 1 mL syringes | Henke-Sass, Wolf | 4010.200V0 | NORM-JECT®; supplier: Grainger, Inc. |
| Human red blood cells | Innovative Research | IPLA-WB3 | Tested and found negative by supplier for: HBsAg, HCV, HIV-1, HIV-2, HIV-1Ag or HIV 1-NAT, ALT, and syphilis by FDA-Approved Methods. Because no test methods can guarantee with 100% certainty the absence of an infectious agent, human derived products should be handled as suggested in the U.S. Department of Health and Human Services Manual on BIOSAFETY IN MICROBIOLOGICAL AND BIOMEDICAL LABORATORIES, FOR POTENTIALLY INFECTIOUS HUMAN SERUM OR BLOOD SPECIMENS |
| Phosphate buffered saline concentrate | P5493 | SIGMA | 10x; diluted to 1x |
| Tween | P9416 | SIGMA | TWEEN® 20 |
| Centrifuge | LW Scientific | STRAIGHT8-5K | Swing-Out 8-place Centrifuge. Available through authorized dealers. Other centrifuges available direct from LW Scientific. |
| HD video recorder | Sony | MHS-CM5 | |
| Orange fluorescent nucleic acid stain | Invitrogen | S-11364 | SYTO® 83 Orange Fluorescent Nucleic Acid Stain. Stored in DMSO solvent. Always wear reccommended Personal Protective Equipment. No special handling advice required. |
| Fluorescent counting beads | Invitrogen | MP 36950 | CountBright™ Absolute Counting Beads. Always wear reccommended Personal Protective Equipment. No special handling advice required. |
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Cite This Article
Phipps, W. S., Yin, Z., Bae, C., Sharpe, J. Z., Bishara, A. M., Nelson, E. S., Weaver, A. S., Brown, D., McKay, T. L., Griffin, D., Chan, E. Y. Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology. J. Vis. Exp. (93), e51743, doi:10.3791/51743 (2014).