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。
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。
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。
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…
The authors have nothing to disclose.
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。
(±)-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 |