Özet

用于从单一DNA分子大规模平行蛋白质合成的Femtoliter滴体阵列

Published: June 20, 2020
doi:

Özet

该协议的总体目标是在 1 厘米2平面基板上准备超过 100 万个有序、均匀、稳定且生物相容的 femtoliter 液滴,可用于无细胞蛋白质合成。

Abstract

科学仪器的空间分辨率和检测灵敏度的进步使得将小型反应堆应用于生物和化学研究成为可能。为了满足对高性能微反应器的需求,我们开发了一种女性滴阵列(FemDA)装置,并举例说明了它在大规模平行无细胞蛋白合成(CFPS)反应中的应用。使用两步油封式协议,在手指大小的区域内容易产生超过 100 万个均匀液滴。每个液滴都锚定在一个女性升微室,由亲水底部和疏水侧壁组成。混合亲水疏水结构以及专用密封油和表面活性剂对于在女性空间中稳稳地保留女性水溶液而不造成蒸发损失至关重要。Femtoliter 配置和 FemDA 器件的简单结构允许最少的试剂消耗。滴滴反应堆的统一尺寸使大规模定量和时间过程测量令人信服和可靠。FemDA 技术将 CFPS 反应的蛋白质产量与每个滴滴中的 DNA 分子数相关。我们简化了有关器件微制造、女性飞沫形成以及获取和分析微观图像数据的程序。具有优化的低运行成本的详细协议使拥有标准洁净室设施和传统荧光显微镜的每个人都可以使用 FemDA 技术。

Introduction

研究人员使用反应器进行生物/化学反应。为了降低试剂消耗,同时提高工作效率,为缩小反应堆尺寸和增加实验吞吐量作出了重大努力。这两个方面旨在使研究人员从繁重的工作量中解放出来,降低成本,加快研发。从反应量和吞吐量的角度,我们有一个明确的历史路线图,即反应器技术的发展:单烧杯/烧瓶/试管、毫升管、微升管、微升8管条、微升96/384/1536孔板和微流体纳米升/皮升/女性,升反应器1、2、3、4、5、6、6。2,3,4,6,715与过去几十年半导体行业集成电路芯片上的晶体管特性尺寸缩小类似,生物/化学微反应器正在经历体积降低和系统集成。这种小规模工具对基于细胞或无细胞的合成生物学、生物制造和高通量原型制作和筛选898、9、10、11、1210,11,12产生了深远的影响。,本文介绍了我们最近开发独特的滴滴阵列技术的努力,并展示了它在CFPS13中的应用,这是合成生物学和分子筛选社区的基础技术。特别是,我们特意提供优化和低成本的协议,使 FemDA 设备可供所有人使用。小型设备低成本且易于处理的协议将有助于大学的教育目的,并有助于推广技术。

FemDA 在平面玻璃基板上以 106/1厘米2的超高密度准备女性飞沫。我们在玻璃基板上涂上疏水性聚合物CYTOP15,并在预定义位置选择性地蚀刻(去除)CYTOP,在基材上产生微腔阵列。因此,产生的微室由疏水侧壁(CYTOP)和亲水底部(玻璃)组成。当水和油在图案表面连续流动时,水将被捕获并密封到微腔中。亲水疏水结构对于排斥微腔外的水、隔离单个微反应器以及在女性空间内保留微小的水溶液至关重要。该独特的特性已成功应用于制备水中油滴和脂质双层微室16、17。,17与原型装置16相比,我们首先优化了微制造工艺,实现了CYTOP聚合物的完全去除,以及玻璃底部的完全曝光。CYTOP 是一种特殊的氟聚合物,表面张力极低(19 mN/m),比玻璃、塑料和硅胶等传统微反应器材料低。其良好的光学、电气和化学性能已应用于微流体装置18、19、20、21、22、23、24的表面处理。18,19,20,21,22,23,24在FemDA系统中,要对CYTOP表面的油进行良好的润湿,油的表面张力必须低于固体表面25。否则,与固体表面接触的液体油往往会变成球形,而不是扩散到表面。此外,我们发现,一些流行的全氟碳油(例如,3M FC-40)16和氢氟16醚油(例如,3M Novec系列)可以溶解CYTOP,由于CYTOP的形态,这是致命的定量测量,在液滴交叉污染方面是值得怀疑的。幸运的是,我们发现了一种生物相容和环保的油,表面张力降低(< 19 mN/m) 表面张力13。我们还发现了一种新的表面活性剂,可以在选定的油中溶解,在低浓度(0.1%,至少比先前报道的流行26,27)13低1026,27倍。13由此产生的水/油界面可以通过表面活性剂稳定。由于油的蒸发率高,在用油冲洗后,我们应用了另一种生物相容和环保的油来替代第一个密封微腔的油。我们分别将第一种油(ASAHIKLIN AE-3000,0.1 wt % SURFLON S-386)称为”冲油”,第二种油(丰布林 Y25)分别称为”密封油”。

两步油封策略可以在几分钟内实现女性滴层阵列的牢固形成,无需精密的仪器。由于蒸发问题,生产小于皮克升体积28的微反应器一直被认为是具有挑战性的。FemDA通过系统地优化用于制备微反应器/液滴的材料和工艺来解决这个问题。产生的液滴的几个值得注意的特点包括高均匀性(或单分散性)、稳定性和在女性级表上的生物相容性。滴体积的变化系数 (CV) 仅为 3%(没有微图像的晕影校正),是世界上滴管平台中最小的 CV,可确保高度平行和定量测量。女性升液滴稳定至少24小时,在室温下液滴之间无交叉污染,对于可靠的时间过程测量非常有价值。关于生物相容性,我们成功地合成了各种蛋白质,从一个单拷贝模板DNA在女性滴,以前被认为是困难或低效的29,30。,30它值得阐明为什么一些能够在FemDA中合成的蛋白质不能在其他滴系统中合成。FemDA不仅是一个技术进步,而且实现了前所未有的定量测量,可以将蛋白质产量(如滴的荧光强度所反映)与每个滴滴中的模板DNA分子数量相关联。因此,基于FemDA的CFPS液滴荧光强度的直方图显示了离散分布,可以通过相等峰峰间隔的高斯分布总和很好地拟合。此外,含有不同数量DNA分子的液滴发生的概率非常适合泊森分布31。因此,根据离散分布,可以对每滴中不同的蛋白质产量进行标准化。这一关键功能使我们能够将酶活性信息与表观强度分开,而其他微反应器平台尚未提供这些信息。现有的微流体细胞/液滴分拣系统擅长全自动分拣,善于浓缩样品,但有时只能在分析方面32、33,33输出相对宽或长尾直方图。我们的高度定量和生物相容的FemDA系统为微反应器开发领域树立了新的基准和较高的分析标准。

可用于制备液滴的油和表面活性剂仍然非常有限。ASAHIKLIN AE-3000 和 SURFLON S-386 在 FemDA 中建立的组合是水相与石油第13阶段之间物理化学界面不断增长的武器库的新成员。FemDA 中的新界面在物理上稳定、化学惰,并且与多种蛋白质的复杂转录、翻译和翻译后修饰机制相容。找到一种在滴下设置中无法合成的蛋白质会相当有吸引力。此外,试剂的成本节约在女性滴滴系统中比在纳米升和皮升反应器系统35,36,36更明显。特别是,在微流体液滴生成系统中,经常会有大量死体积,这主要是由管道或外部供应造成的,而不是在我们的FemDA中。阵列格式还受到每个反应堆37的重复和详细的微观特征(类似于所谓的高含量分析)的青睐,而不仅仅是快速移动物体的单个快照。女性升表使100多万个反应堆能够整合到手指大小的区域,而同样数量的纳米升反应堆(如果存在的话)需要超过平方米的面积,这无疑不适合制造或使用这种系统。

Protocol

1. 女性微腔阵列基板的微制造 注:在洁净室进行以下微制造实验。进入洁净室前,请戴上手套和洁净室套装。 清洁盖玻璃基板 将盖玻璃放在盖玻璃染色架上。在室温 (RT) 下,将 8 M 氢氧化钠 (NaOH) 中的盖玻璃声波 15 分钟。注意:高浓度的NaOH对皮肤和眼睛非常危险。轻轻处理它,没有任何飞溅。 将机架从 NaOH 溶液中拿出来,用水冲洗盖玻璃十次…

Representative Results

微制造工艺包括基材清洗、表面功能化、CYTOP涂层、光刻、干蚀刻、光刻剥离和最终清洁。重要的是,所提出的协议允许完全去除微腔图3A内的疏水性CYTOP聚合物,在标准盖玻璃基板上产生高度平行的亲水疏水结构。在油封协议的帮助下,通过在微腔中封装荧光溶液,验证了结果液滴的统一尺寸(图3B)。使用开发的软件提取的荧光强度是滴径的良好指?…

Discussion

基于 FemDA 中高度均匀、稳定和生物相容液滴的高定量测量使离散分布得以实现,这是我们研究的独特特征,不同于其他。本文系统地优化并详细介绍了微制造和滴层形成过程。已建立的协议中有几个关键步骤。

首先,在矩形薄玻璃基板上高粘性CYTOP聚合物的统一涂层在很大程度上决定了结果基板的质量。鉴于目标透镜的工作距离通常较短,放大倍率高(参见步骤 6.1),必须?…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

这项工作得到了JSPS KAKENHI赠款号JP18K14260和日本海洋-地球科学技术厅预算的支持。我们感谢德古奇(JAMSTEC)和伊库塔(JAMSTEC)提供特征设施。我们感谢 Ken Takai (JAMSTEC) 的商业软件支持。微制造在东京大学武田森田超洁净室进行,由日本教育文化、体育、科学技术省(MEXT)的”纳米技术平台计划”支持,授予编号JPMXP09F19UT0087。

Materials

(3-aminopropyl)triethoxysilane Sigma-Aldrich 440140
1 mL syringe Terumo SS-01T
2-propanol Kanto Chemical EL grade EL: for electronic use.
3D laser scanning confocal microscope Lasertec OPTELICS HYBRID Other similar microscopes (e.g., Keyence VK-X1000, Olympus LEXT OLS5000) are also applicable.
50 mL syringe Terumo SS-50LZ
6,8-difluoro-4-methylumbelliferyl phosphate Thermo Fisher Scientific D6567 Prepare a 5 mM stock solution in dimethyl sulfoxide
Acetone Kanto Chemical EL grade EL: for electronic use.
Purity 99.8%.
Air blower Hozan Z-263
Aluminum block BIO-BIK AB-24M-02
Aluminum microtube stand BIO-BIK AB-136C
ASAHIKLIN AE-3000 AGC (Test sample) Free test sample may be available upon inquiry to AGC.
BEMCOT PS-2 wiper Ozu 028208
Biopsy punch with plunger Kai BPP-10F
Cover glass Matsunami Glass No. 1 (24 mm × 32 mm, 0.13~0.17 mm thickness) Size-customized.
Cover glass staining rack Nakayama 803-131-11
CRECIA TechnoWipe clean wiper Nippon Paper Crecia C100-M
Cutting mat GE Healthcare WB100020
CYTOP AGC CTL-816AP
Deaeration mixer Thinky AR-100
Desktop cutter Roland STIKA SV-8
Developer AZ Electronic Materials AZ 300 MIF AZ Electronic Materials was now acquired by Merck.
Other alkaline developers may be also applicable but should require optimization of development conditions (time, temperature, etc.)
Double-coated adhesive Kapton film tape Teraoka Seisakusho 7602 #25
Ethanol Kanto Chemical EL grade EL: for electronic use.
Purity 99.5%.
Fiji Version: ImageJ 1.51n
Flat-cable cutter Tokyo-IDEAL MT-0100
Fomblin oil Solvay Y25, or Y25/6 Free test sample may be available upon inquiry to Solvay. Fomblin Y25/6 is an alternative if Y25 is not readily available.
Hot plate AS ONE TH-900
Injection needle Terumo NN-2270C 22G × 70 mm
Inverted fluorescence microscope Nikon Eclipse Ti-E Epifluorescence specification, CCD or sCMOS camera, motorized stage, autofocus system, and high NA objective lens are required.
KaleidaGraph Synergy Version: 4.5
Mask aligner SUSS MA-6 Other mask aligners are also applicable as long as the vacuum contact mode is avaliable.
MICROMAN pipette GILSON E M250E Capillary piston tip: CP250
Microsoft Excel Microsoft Version: 16.16.15
Mini vacuum chamber AS ONE MVP-100MV
Nuclease-free water NIPPON GENE 316-90101
Parafilm Amcor PM-996
PCR tube NIPPON Genetics FG-021D/SP
Petri dish AS ONE GD90-15 Diameter 90 mm, height 15 mm.
Photoresist AZ Electronic Materials AZ P4903 AZ Electronic Materials was now acquired by Merck. AZ P4620 is an alternative.
Plate reader BioTek POWERSCAN HT
Polyethelene gloves AS ONE 6-896-02 Trade name: Saniment.
PURExpress in vitro protein synthesis kit New England Biolabs E6800S or E6800L For cell-free protein synthesis reaction.
Reactive-ion etching system Samco RIE-10NR Other RIE systems are also applicable but should require optimization of RIE conditions (gas flow rate, chamber pressure, RF power, etching time, etc.)
RNase inhibitor New England Biolabs M0314S
Scotch tape 3M 810-1-18D
Sodium hydroxide solution FUJIFILM Wako Pure Chemical 194-09575 8 M concentration; danger.
Spin coater Oshigane SC-308
SURFLON S-386 surfactant AGC (Test sample) Free test sample may be available upon inquiry to AGC.
SYLGARD 184 silicone elastomer Dow Sylgard184 Chemical composition: polydimethylsiloxane. The default mixing ratio is base : curing agent = 10 : 1 (m/m).
Tweezers Ideal-tek 2WF.SA.1
2A
Ultrasonic cleaner AS ONE ASU-2M
Vacuum chuck Oshigane (Customized) Material: delrin; rectangular sample stage with multiple holes (48 holes, each with 1 mm diameter); the size is customzied to fit the size of the cover glass (24 mm × 32 mm).

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Zhang, Y., Kurosawa, K., Nishiura, D., Tei, M., Tsudome, M. A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules. J. Vis. Exp. (160), e60945, doi:10.3791/60945 (2020).

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