JoVE Journal > Engineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Engineering

使用温度控制基于微管的3D活性流体的流速

Published: November 26, 2019
doi:
10.3791/60484
, , ,
1Department of Physics,Worcester Polytechnic Institute, 2Department of Physics,Brandeis University

Summary

该协议的目的是使用温度来控制三维有源流体的流动速度。这种方法的优点不仅允许原地调节流速，还可实现动态控制，例如定期调优流速。

Abstract

我们提出了一种利用温度来调整运动因子驱动的基于微管的三维（3D）有源流体的流速的方法。此方法允许原地调整速度，而无需制造新样品以达到不同的期望速度。此外，该方法可实现速度的动态控制。循环温度会导致流体周期性地快速流动和缓慢流动。这种可控性基于激酶-微管反应的Arrhenius特性，显示了4~8μm/s的受控平均流速范围。提出的方法将为微流体器件的设计打开大门，在微流体器件中，通道中的流速可局部调节，无需阀门。

Introduction

主动物质与传统无源物质不同，因为能将化学能转化为机械工作。具有这种能力的材料可以包括活的或非生物的实体，如细菌、昆虫、胶体、谷物和细胞骨骼细丝1，2，3，4，5，6，7，8，9，10。这些物质实体与其邻居交互。在更大的尺度上，它们自组织成湍状涡流（主动湍流）或物质流动11，12，13，14，15，16，17，18，19，20。对活性物质自组织的理解已导致分子穿梭、光学器件和并行计算21、22、23的各种应用。要将应用程序带到一个新的水平，需要超越自组织的控制。例如，Palacci等人研制出一种赤铁矿封装的胶体，只有在暴露在手动控制的蓝光下才能自行推进，这导致了活晶体24的出现。Morin等人利用可调谐的外部电场建立了滚动昆克胶体的控制，导致胶体在赛道状的通道25中形成。这些以前的工作展示了局部控制在应用中的作用，并推进了活性物质的知识库。

在本文中，我们重点介绍了运动因子驱动的基于微管 （MT） 的 3D 活性流体的可控性。流体由三个主要成分组成：MT、激酶分子马达和消耗物。消耗物会产生消耗力来捆绑 MT，这些 MT 后来被电机簇桥接。这些电机沿着 MT 向加端移动。当一对桥接的MTsis反平行时，相应的电机朝相反的方向行走。但是，电机被绑定在一个簇中，无法分开，因此它们协同滑动成对的 MT（间丝滑动，图 1 A）。这些滑动动力学积累，导致束的MTsto延伸，直到达到其屈曲不稳定点和断裂（扩展束，图1B）26。破碎的束被耗尽力退火，随后再次延伸，动态重复。在重复动力学过程中，捆绑运动搅动附近的液体，诱导流动，可以通过使用微米级示踪剂进行掺平来可视化（图1C）。桑切斯等人和Henkin等人对示踪剂的平均速度进行了描述，发现通过改变三磷酸腺苷（ATP）、消耗物、电机簇和MC19、27的浓度，速度是可调节的。然而，这种可调谐性仅在活性流体合成之前存在。合成后，适应性丧失，流体以自己的方式自行组织。为了控制合成后的活性流体活性，Ross.etal.报告了一种使用光激活的马达蛋白二聚化方法，允许使用光28调节流体活性。虽然光控制在局部激活流体方面是方便的，但该方法需要重新设计运动蛋白的结构，同时在显微镜中修改光学路径。在这里，我们提供了一种易于使用的方法，用于在无需显微镜修改的情况下控制流体流动，同时保持电机结构不变。

我们的局部调谐有源流体流动的方法基于阿雷尼乌斯定律，因为激酶-MT反应已经报告随着温度29，30，31，32增加。我们先前的研究表明，活性流体流的平均速度的温度依赖性遵循Arrhenius方程：v = A exp（- Ea/RT），其中A是指数前因子，R是气体常数，Ea是活化能，T是系统温度33。因此，流体活性对温度环境敏感，系统温度需要一致，以稳定电机性能，因此，流体流动速度为34。在本文中，我们演示了利用电机的温度依赖性通过调整系统温度来持续调整有源流体的流速。我们还演示了活性流体样品的制备，然后将样品安装在通过计算机软件控制温度的显微镜阶段。将温度从16°C提高到36°C可加快平均流速从4至8μm/s。 此外，可调节性是可逆的：反复升高和降低温度顺序加速和减速流。演示的方法适用于主要反应遵循Arrhenius定律的各种系统，如MT滑翔测定29、30、31、32。

Protocol

1. 编制MT 注意：在这一步中，我们从牛脑组织中纯化牛头蛋白。牛脑可能导致变种克罗伊茨费尔特-雅科布病（vCJD）35。因此，应将脑废物及相关溶液、瓶子和移液器吸头收集在生物垃圾袋中，并按照机构规定作为生物危险废物进行处理。 从牛脑中纯化牛头蛋白（从Castoldi等人改过自36年）。 将大约1.5公斤的新鲜牛脑从当地的?…

Representative Results

准备运动动酶驱动的基于MT的有源流体需要运动因子和MT。MC 从标记的 tubulin（步骤 1.3 和 1.4）中聚合，这些牛脑被纯化（步骤 1.1，图 2A），然后回收以提高纯度（步骤 1.2，图 2B）。激酶运动蛋白在大肠杆菌中表达和纯化（步骤2.1和2.2，图2B）41，52。 <str…

Discussion

就地控制活性物质为主动物质4、5、24、28、54的定向自组织打开了大门。在本文中，我们提出了一个协议，使用温度来控制运动因子驱动的，基于MT的有源流体的原位，基于系统的Arrhenius特性29，30，31。由…

Disclosures

The authors have nothing to disclose.

Acknowledgements

疟原虫K401-BCCP-H6是兹沃尼米尔·多吉奇博士的礼物。这项研究得到了吴坤达博士在伍斯特理工学院的创业基金的支持。我们感谢Zvonimir Dogic博士为净化和标记图布林和合成活性液体的协议。我们感谢马克·里迪拉博士在蛋白质表达和纯化方面的专长。我们感谢威廉·本杰明·罗杰博士协助我们建立温控阶段。我们确认布兰代斯 MRSEC （NSF-MRSEC-1420382） 使用生物材料设施 （BMF）。我们感谢英国皇家化学学会对贝特等人关于软物质33的数据进行了调整。

Materials

(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid Sigma-Aldrich 238813 Trolox
2-Mercaptoethanol Sigma-Aldrich M6250
3-(Trimethoxysilyl)propyl methacrylate, 98%, ACROS Organics Fisher Scientific AC216550050
3.2mm I.D. Tygon Tubing R-3603 HACH 2074038 Water tubes
31.75 mm diameter uncoated, sapphire window Edmund Optics 43-637 Sapphire disc
3M 1181 Copper Tape – 1/2 IN Width X 18 YD Length – 2.6 MIL Total Thickness – 27551 R.S. HUGHES 054007-27551 Copper tape
Acetic Acid Sigma-Aldrich A6283
Acrylamide Solution (40%/Electrophoresis), Fisher BioReagents Fisher Scientific BP1402-1
Adenosine 5'-triphosphate dipotassium salt hydrate Sigma-Aldrich A8937 ATP
Alexa Fluor 647 NHS Ester (Succinimidyl Ester) Thermo Fisher Scientific A20006 Far-red fluorescent dye. Alexa 647 can be pre suspended in dimethylsulfoxide (DMSO) before mixing with microtubules (1.3.3.2.)
Amicon Ultra-4 Centrifugal Filter Unit Sigma-Aldrich UFC801024 Centrifugal filter tube. Cutoff molecular weight: 10 kDa
Ammonium Persulfate, 100g, MP Biomedicals Fisher Scientific ICN802829 APS
Ampicillin Sodium Salt (Crystalline Powder), Fisher BioReagents Fisher Scientific BP1760 Ampicillin
Antivibration Table Nikon 63-7590S
Avanti J-E Centrifuge Beckman Coulter 369001
Bacto Agar Soldifying Agent, BD Diagnostics VWR 90000-760 Agar
Biotin Alfa Aesar A14207
Bucket-plastic white – 2 gallon Bon 84-715 Water bucket
Calcium Chloride Sigma-Aldrich 746495 CaCl2
Catalase from bovine liver Sigma-Aldrich C40
CFI Plan Apo Lambda 4x Obj Nikon MRD00045 4x air objective
C-FLLL-FOV GFP HC HC HISN ero Shift Nikon 96372 GFP filter cube
CH-109-1.4-1.5 TE Technology CH-109-1.4-1.5 Thermoelectric Cooler (TEC)
Chloramphenicol, 98%, ACROS Organics Fisher Scientific C0378
Cooling block N/A N/A Custom milled aluminum
Coomassie Brilliant Blue R-250 #1610400 Bio-Rad 1610400 Triphenylmethane dye
D-(+)-Glucose Sigma-Aldrich G7528
Dimethyl Sulfoxide (Certified ACS), Fisher Chemical Fisher Scientific D128 DMSO
DL-1,4-Dithiothreitol, 99%, for biochemistry, ACROS Organics Fisher Scientific AC165680050 DTT
DOWSIL 340 Heat Sink Compound Dow 1446622 Thermal paste
ETHYL ALCOHOL, 200 PROOF ACS/USP/NF GRADE 5 GALLON POLY CUBE Pharmco by Greenfield Global 111000200CB05 Ethanol
Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid Sigma-Aldrich E3889 EGTA
Ethylenediaminetetraacetic acid Sigma-Aldrich 798681 EDTA
Fisher BioReagents Microbiology Media Additives: Tryptone Fisher Scientific BP1421 Tryptone
Fisher BioReagents Microbiology Media Additives: Yeast Extract Fisher Scientific BP1422 Yeast extract
Fluoresbrite YG Microspheres, Calibration Grade 3.00 µm Polysciences 18861 Tracer particles
Glucose Oxidase from Aspergillus niger Sigma-Aldrich G2133
Glycerol Sigma-Aldrich G5516
GpCpp Jena Bioscience NU-405L Guanosine-5′[(α,β)-methyleno]triphosphate (GMPCPP)
GS Power's 18 Gauge (True American Wire Ga), 100 feet, 99.9% Stranded Oxygen Free Copper OFC, Red/Black 2 Conductor Bonded Zip Cord Power/Speaker Electrical Cable for Car, Audio, Home Theater Amazon B07428NBCW Copper wire
Guanosine 5'-triphosphate sodium salt hydrate Sigma-Aldrich G8877 GTP
Hellmanex III Sigma-Aldrich Z805939 Detergent
HEPES Sodium Salt (White Powder), Fisher BioReagents Fisher Scientific BP410 NaHEPES
High performance blender machine AIMORES AS-UP1250 Blender
His GraviTrap GE Healthcare 11003399 Gravity Column
Imidazole Sigma-Aldrich I5513
IPTG Sigma-Aldrich I6758 Isopropyl β-D-1-thiogalactopyranoside
Isopropyl Alcohol 99% Pharmco by Greenfield Global 231000099 Isopropanol
JA-10 rotor Beckman Coulter 369687
L-Glutamic acid potassium salt monohydrate Sigma-Aldrich G1501 K-Glutamate
Lysozyme from chicken egg white Sigma-Aldrich L6876
Magnesium chloride hexahydrate Sigma-Aldrich M2670 MgCl2•6H2O
MES sodium salt Sigma-Aldrich M5057 2-(N-Morpholino)ethanesulfonic acid sodium salt
MOPS Sigma-Aldrich M1254 3-(N-Morpholino)propanesulfonic acid
MP-3022 TE Technology MP-3022 Thermocouple
N,N,N',N'-Tetramethylethylenediamine 99%, ACROS Organics Fisher Scientific AC138450500 TEMED
Nanodrop 2000c UV-VIS Spectrophotometer Thermo Fisher Scientific E112352 Spectrometer
Nikon Ti2-E Nikon Inverted Microscope Nikon MEA54000
Norland Optical Adhesive 81 Norland Products NOA81 UV glue
Novex Sharp Pre-stained Protein Standard Thermo Fisher Scientific LC5800 Protein standard ladder
NuPAGE 4-12% Bis-Tris Protein Gels, 1.5 mm, 10-well Thermo Fisher Scientific NP0335BOX SDS gel
Optima L-90K Ultracentrifuge Beckman Coulter 365672
Parafilm PM996 Wrap , 4" Wide; 125 Ft/Roll Cole-Parmer EW-06720-40 Wax film
Pe 300 ultra Illumination System Single
Band , 3mm Light Guide control Pod
power supply		 Nikon PE-300-UT-L-SB-40 Cool LED Illuminator
Phenylmethanesulfonyl fluoride Sigma-Aldrich 78830 PMSF
Phosphoenolpyruvic acid monopotassium salt, 99% BeanTown Chemical 129745 PEP
Pierce Coomassie (Bradford) Protein Assay Kit Thermo Fisher Scientific 23200
Pierce Protease Inhibitor Mini Tablets Thermo Fisher Scientific A32953
PIPES Sigma-Aldrich P6757 1,4-Piperazinediethanesulfonic acid
Pluronic F-127 Sigma-Aldrich P2443
Poly(ethylene glycol) Sigma-Aldrich 81300 PEG. Average molecular weight 20,000 Da
Potassium Hydroxide (Pellets/Certified ACS), Fisher Chemical Fisher Scientific P250-500 KOH
PowerEase 300W Power Supply (115 VAC) ThermoFisher Scientific PS0300 DC power supply of the gel box
PS-12-8.4A TE Technology PS-12-8.4A DC power supply of the temperature controller
Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle Sigma-Aldrich P-0294 PK/LDH
Quiet One Lifegard Fountain Pump, 296-Gallon Per Hour Amazon B005JWA612 Fish tank pump
Rosetta 2(DE3)pLysS Competent Cells – Novagen Millipore Sigma 71403 Competent cells
Sharp Microwave ZSMC0912BS Sharp 900W Countertop Microwave Oven, 0.9 Cubic Foot, Stainless Steel Amazon B01MT6JZMR Microwave for boiling the water
Sodium Chloride (Crystalline/Certified ACS), Fisher Chemical Fisher Scientific S271-500 NaCl
Sodium dodecyl sulfate Sigma-Aldrich L3771 SDS
Sodium phosphate monobasic Sigma-Aldrich S8282 NaH2PO4
Streptavidin Protein Thermo Fisher Scientific 21122
Sucrose Sigma-Aldrich S7903
TC-720 TE Technology TC-720 Temperature controller
Tris Base, Molecular Biology Grade – CAS 77-86-1 – Calbiochem Sigma-Aldrich 648310 Tris-HCL
Type 45 Ti rotor Beckman Coulter 339160
Type 70 Ti rotor Beckman Coulter 337922
Type 70.1 Ti rotor Beckman Coulter 342184
VWR General-Purpose Laboratory Labeling Tape VWR 89097-916 Paper tapes
VWR Micro Cover Glasses, Square, No. 1 1/2 VWR 48366-227 Glass coverslips
VWR Plain and Frosted Micro Slides, Premium VWR 75799-268 Glass slides
XCell SureLock Mini-Cell ThermoFisher Scientific EI0001 Gel box
ZYLA 5.5 USB3.0 Camera Nikon ZYLA5.5-USB3 Monochrome CCD camera
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MicrotubuleActive FluidTemperature ControlKinesinATPPolyethylene GlycolPhoto-bleachingTracer ParticlesPolyacrylamideGlass Slide

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Bate, T. E., Jarvis, E. J., Varney, M. E., Wu, K. Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature. J. Vis. Exp. (153), e60484, doi:10.3791/60484 (2019).
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