机械节点孔传感：用于多参数单细胞粘弹性测量的快速、无标记平台
Summary
这里介绍的是一种使用称为机械节点孔传感（mechano-NPS）的基于电子学的微流体平台对单个细胞进行机械表型分析的方法。该平台保持1-10个细胞/秒的中等通量，同时测量细胞的弹性和粘性生物物理特性。
Abstract
细胞力学特性涉及各种生物过程和疾病，从干细胞分化到癌症转移。测量这些特性的常规方法，如原子力显微镜（AFM）和微量移液器抽吸（MA），捕获了丰富的信息，反映了细胞的全粘弹性响应;但是，这些方法受到非常低的吞吐量的限制。实时变形性细胞术（RT-DC）等高通量方法只能测量有限的机械信息，因为它们通常仅限于仅反映细胞弹性特性的单参数读数。与这些方法相比，机械节点孔传感（mechano-NPS）是一种灵活的无标记微流体平台，可在实现中等通量的细胞的多参数粘弹性测量方面弥合差距。直流 （DC） 测量用于监测细胞通过微流体通道时，跟踪它们在被迫通过狭窄收缩之前、期间和之后的大小和速度。该信息（即大小和速度）用于量化每个细胞的横向变形、抗变形能力和变形恢复。通常，这种基于电子学的微流体平台提供多种粘弹性电池特性，从而更完整地了解电池的机械状态。由于它需要最少的样品制备，采用简单的电子测量（与高速相机相比），并利用标准的软光刻制造，因此该平台的实施简单易用，并且适用于下游分析。该平台的灵活性、实用性和灵敏度为各种细胞提供了独特的机械信息，在基础科学和临床诊断中具有更多应用的潜力。
Introduction
单细胞是动态的粘弹性材料1。许多内部和外部过程（例如，有丝分裂的发作或细胞外基质[ECM]的重塑）影响其结构和组成2，3，4，通常导致补充其当前状态的独特生物物理特性。特别是，机械性能已被证明是细胞发育，生理学和病理学的重要生物标志物，产生有价值的定量信息，可以补充规范的分子和遗传方法5，6，7。例如，Li等人最近描述了耐药性和药物反应性急性早幼粒细胞白血病细胞之间的机械差异，同时还使用RNA-seq揭示了差异表达的细胞骨架相关基因8。通过了解单细胞力学和细胞功能之间的复杂相互作用，机械分型在改变基础科学和临床诊断方面具有更广泛的应用9。
测量单细胞力学的最广泛采用的工具是原子力显微镜（AFM）。虽然AFM能够对细胞机械特性进行高分辨率的局部测量，但它仍然局限于<0.01个细胞/秒10的通量。或者，光学拉伸器使用两个发散的激光束来捕获和变形悬浮的单个细胞11，仅限于略高的吞吐量<1细胞/秒12。微流体技术的最新进展使新一代设备能够进行快速，单细胞，机械评估12，13。这些技术采用窄收缩通道14，15，剪切流16或流体动力拉伸17，以10-1，000个细胞/秒的吞吐量快速变形细胞18。虽然这些方法的测量速度比传统技术快得多，但它们经常以高通量能力换取有限的机械读数（补充表1）。所有上述快速微流体方法都侧重于基本的单参数指标，例如传输时间或变形率，这些指标仅反映细胞的弹性特性。然而，鉴于单细胞的内在粘弹性，对细胞进行稳健而彻底的机械表征不仅需要考虑弹性成分，还需要考虑粘性响应。
机械节点孔传感（机械NPS）2，8（图1A）是一种微流体平台，可解决单细胞机械化存在的局限性。该方法可以同时测量多个生物物理参数，包括细胞直径、相对变形性和变形恢复时间，吞吐量适中，为 1-10 个细胞/秒。该技术基于节点孔传感（NPS）19，20，21，22，23，24，它涉及使用四点探针测量来测量细胞通过微流体通道产生的调制电流脉冲，该通道已被更广泛的区域（称为“节点”）分割。调制电流脉冲是电池部分阻塞段（即“孔隙”）和节点中的电流的结果，前者比后者阻挡的电流更多。在机械NPS中，一个片段，即“收缩通道”，比细胞直径窄;因此，细胞必须变形才能通过整个通道（图1B）。细胞直径可以通过细胞在收缩通道之前通过节点孔时产生的亚脉冲的大小来确定（图1B，C）。这里，|ΔInp|，当细胞在孔中时的电流下降，与细胞与孔的体积比成正比，V细胞/V孔2，8，19。细胞刚度可以通过Δ T c来确定，ΔTc是细胞通过收缩通道时产生的明显较大的亚脉冲的持续时间（图1B，C）。较硬的细胞比较软的细胞需要更长的时间来通过通道2，8。最后，细胞“恢复”，即细胞在变形后恢复到其原始大小和形状的能力，可以通过细胞在收缩通道后通过节点孔时产生的一系列亚脉冲来确定（图1B，C）。恢复时间ΔTr是在电池被挤压之前电流子脉冲恢复到先前子脉冲幅度所需的时间。总体而言，记录和分析电池通过微流体通道时产生的调制电流脉冲，以提取相关的单电池机械参数（图1D）2，8。
这种基于电子的微流体平台的可重复性和易用性先前已得到证明25。此外，该平台为单细胞机械化提供了较低的进入门槛。采用标准软光刻技术来制造微流体器件。测量 硬件 由 廉价 的 组 件 组成， 包括 简单 的 印刷 电路板 （PCB）、 电源、 前 置 放大器、 数据 采集 板 （DAQ） 和 计算机。最后，用户友好的代码可用于数据采集和分析，从而实现直接实施。这种机械分型技术可以区分非恶性和恶性乳肺上皮细胞系的群体，区分原代人乳腺上皮细胞的亚谱系，并表征细胞骨架扰动和其他药理学因素的影响2，8。总体而言，该平台是单细胞机械分型的有效方法。
Protocol
1. 设计设备几何形状 选择大小调整和恢复段的宽度，使其宽于要测量的最大单元的直径，同时保持足够的信噪比（SNR）。有关各种细胞系的不同大小和回收段宽度的示例，请参阅 补充表2 。 选择收缩段宽度以将30%-40%的应变施加到要进行机械化处理的细胞的平均大小上。应变定义为 ，其中 <…
Representative Results
这里介绍的机械分型平台是一种简单而通用的方法，用于测量具有中等通量的单个细胞的生物物理特性。使用恒压驱动流将细胞流过微流体通道（图1A）。当细胞过境时，使用数据采集硬件记录微流体通道的长度和产生的电流脉冲。然后使用MATLAB上的自定义软件处理采集的信号（图1B，C），以提取相关的单电池机械性能。 ?…
Discussion
使用这种机械分型技术测量单细胞的机械性能包括三个阶段：设备制造、数据采集和数据分析。在每个阶段中，都有可能显着影响实验结果的显着方面。在器件制造过程中，一致的通道几何形状和器件间均匀性对于准确和可重复的结果至关重要。具体来说，每个设备的侧壁应该相对光滑（图4Ai），并且复制设备的通道高度应该相当。任何具有部分阻碍细胞流动的缺陷的装置?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了NIBIB 1R01EB024989-01和NCI 1R01CA190843-01的资助。A.L.和R.R.得到了H2H8协会研究生研究奖学金的支持。K. L. C. 得到了美国国家科学基金会研究生研究奖学金和 Siebel 学者奖学金的支持。
Materials
| Acetone | J.T. Baker | 5356-05 | Purity (GC) ≥ 99.5% (https://us.vwr.com/store/product/6057739/acetone-99-5-vlsi-j-t-baker) |
| Aluminum Foil | n/a | n/a | |
| Analog Low-Pass Filter | ThorLabs | EF504 | ≤240 kHz Passband, Coaxial BNC Feedthrough (https://www.thorlabs.com/thorproduct.cfm?partnumber=EF504#ad-image-0) |
| Biopsy Punch | Integra Miltex | 33-31AA-P/25 | 1mm, Disposable, with Plunger (https://mms.mckesson.com/product/573313/Miltex-33-31AA-P25) |
| Blade | n/a | n/a | |
| BNC Cable | Pomona Electronics | 2249-C-12 | https://www.digikey.com/en/products/detail/pomona-electronics/2249-C-12/603323?utm_adgroup=Coaxial%20Cables%20%28RF%29&utm_source=google&utm_ medium=cpc&utm_campaign= Shopping_Product_Cable%20Assemblies_NEW&utm_term= &utm_content=Coaxial%20Cables%20%28RF%29&gclid=Cj0KCQjwlK-WBhDjARIsAO2sErQqnVJ pj5OXVObuTI8ZUf1ZeIn7zvzGnx mCWdePrG6SdEJMF3X6ubUaAs w-EALw_wcB |
| Cleanroom Polyester Swab | Thermo Fisher Scientific | 18383 | https://www.fishersci.com/shop/products/texwipe-cleantip-alpha-polyester-series-swabs-6/18383 |
| Current Preamplifier | DL Instruments | 1211 | https://www.brltest.com/index.php?main_page=product_info&products_ id=1419 |
| Custom PCB (w/ components) | n/a | n/a | see Supplemental files 4 and 5 |
| DAQ Terminal Block | National Instruments | BNC-2120 | https://www.ni.com/en-in/support/model.bnc-2120.html |
| DAQ to BNC-2110 cable | National Instruments | SHC68-68-EPM | https://www.ni.com/en-in/support/model.shc68-68-epm.html |
| Data Acquisition Board (DAQ) | National Instruments | PCI-6251 | https://www.ni.com/docs/en-US/bundle/pci-6251-feature/page/overview.html |
| Dessicator | Thermo Fisher Scientific | 5311-0250 | https://www.thermofisher.com/order/catalog/product/5311-0250 |
| Female BNC To Banana Plug Adapter | Pomona Electronics | 72909 | https://www.digikey.com/en/products/detail/pomona-electronics/72909/1196318 |
| Fetal Bovine Serum (FBS) | VWR | 89510-186 | https://us.vwr.com/store/product/18706419/avantor-seradigm-select-grade-usda-approved-origin-fetal-bovine-serum-fbs |
| Glass Cutter | Chemglass | CG-1179-21 | https://chemglass.com/plate-glass-cutters-diamond-tips |
| Gold Etchant TFA | Transene | NC0977944 | https://www.fishersci.com/shop/products/NC0977944/NC0977944 |
| Hot Plate | Thermo Fisher Scientific | SP131825 | |
| Isopropyl Alcohol | Spectrum Chemical | I1056-4LTPL | Purity (GC) ≥99.5% (https://www.spectrumchemical.com/isopropyl-alcohol-99-percent-fcc-i1056) |
| Metal Hardware Enclosure | Hammond Manufacturing | EJ12126 | https://www.digikey.com/en/products/detail/hammond-manufacturing/EJ12126/2423415 |
| Methanol | Sigma-Aldrich | 34860 | Purity (GC) ≥99.8% (https://www.sigmaaldrich.com/IN/en/substance/methanol320467561) |
| MF-321 Developer | Kayaku Advanced Materials | n/a | https://kayakuam.com/products/mf-321/ |
| MICROPOSIT S1813 Positive Photoresist | DuPont | n/a | https://kayakuam.com/products/microposit-s1800-g2-series-photoresists/ |
| Phosphate Buffered Saline (PBS) | Thermo Fisher Scientific | 10010049 | https://www.thermofisher.com/order/catalog/product/10010049?SID=srch-hj-10010049 |
| Photomask | Fineline Imaging | n/a | Photomask are custom ordered from our CAD designs (https://www.fineline-imaging.com/) |
| Plain Glass Microscope Slide | Fisher Scientific | 12-553-5B | Material: Soda Lime, L75 x W50 mm, Thickness: 0.90–1.10 mm |
| Plasma Cleaner | Harrick Plasma | PDC-001 | https://harrickplasma.com/plasma-cleaners/expanded-plasma-cleaner/ |
| Plastic Petri Dish | Thermo Fisher Scientific | FB0875712 | 100 mm (https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-raised-ridge-100-x-15mm/FB0875712) |
| Pressure Controller | Fluigent | MFCS-EZ | https://www.fluigent.com/research/instruments/pressure-flow-controllers/mfcs-series/ |
| Pressure Controller Software | Fluigent | MAESFLO | |
| Programming & Computation Software | MATLAB | R2021b | for data acquisition and analysis (https://www.mathworks.com/products/matlab.html) |
| PTFE Tubing | Cole Parmer | 06417-31 | 0.032" ID x 0.056" (https://www.coleparmer.com/i/masterflex-transfer-tubing-microbore-ptfe-0-032-id-x-0-056-od-100-ft-roll/0641731) |
| Scepter 2.0 Handheld Automatic Cell Counter | Millapore Sigma | PHCC20060 | https://www.sigmaaldrich.com/IN/en/product/mm/phcc20060 |
| Silicon Wafer | Wafer World | 2885 | 76.2 mm, Single Side Polished (https://www.waferworld.com/product/2885) |
| Spin Coater | n/a | n/a | |
| SU-8 3025 Negative Photoresist | Kayaku Advanced Materials | n/a | https://kayakuam.com/products/su-8-2000/ |
| SU8 Developer | Kayaku Advanced Materials | n/a | https://kayakuam.com/products/su-8-developer/ |
| Sygard 184 Polydimethlysiloxane | Dow Chemical | 4019862 | https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-184-silicone-encapsulant-clear-0.5-kg-kit/ |
| Tape | Scotch | 810-341296 | https://www.staples.com/Scotch-Magic-Tape-810-3-4-x-36-yds-1-Core/product_130567?cid=PS:GS:SBD:PLA:OS&gclid= Cj0KCQjwlK-WBhDjARIsAO 2sErRwzrrgjU0NjFkDkne1xm vT7ekS3tdzvAgiMDwPoxocgH VTQZi7vJgaAvQZEALw_wcB |
| Titanium, Platinum, Gold | n/a | n/a | |
| Triple Output Power Supply | Keysight | E36311A | https://www.newark.com/keysight-technologies/e36311a/dc-power-supply-3o-p-6v-5a-prog/dp/15AC9653 |
| UV Mask Aligner | Karl Suss America | MJB3 Mask Aligner |
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Cite This Article
Lai, A., Rex, R., Cotner, K. L., Dong, A., Lustig, M., Sohn, L. L. Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements. J. Vis. Exp. (190), e64665, doi:10.3791/64665 (2022).