Summary

生成受控动态化学景观,研究微生物行为

Published: January 31, 2020
doi:

Summary

提出了在微流体和微流体设置中通过光液生成动态化学景观的协议。该方法适用于研究不同的生物过程,包括单细胞和种群层面的移动行为、营养摄入或对微生物化学物质的适应。

Abstract

我们演示了一种生成受控动态化学脉冲的方法,即局部化学吸引剂在微观尺度上突然出现,为微生物化学实验创造微环境。为了产生化学脉冲,我们开发了一个系统,通过在含有细菌悬浮液的聚二甲基硅氧烷(PDMS)微流体室中对笼体氨基酸进行光解,在近乎瞬间引入氨基酸源。我们应用了这种方法的化学细菌,Vibrio ordalii,它可以积极爬上这些动态化学梯度,同时通过视频显微镜跟踪。氨基酸,通过化学改性与可拆卸保护组进行化学修饰,在悬浮液中均匀存在,但在它们突然释放之前不能食用,这种释放通过近UV-A聚焦LED光束在用户定义的时间和空间点发生。脉冲中释放的分子数量可以通过暴露时间和未处理分数之间的校准关系来确定,其中光解后的吸收光谱使用 UV-Vis 光谱进行特征。纳米多孔聚碳酸酯 (PCTE) 膜可集成到微流体装置中,以便通过流动连续去除未结折合的化合物和废介质。PCTE膜与PDMS微流体结构之间的强、不可逆的粘结,是通过用3-氨基丙烯氧硅烷(APTES)溶液涂覆膜,然后对要粘结的表面进行等离子体活化来实现的。计算机控制的系统可以在不同位置和不同强度下生成用户定义的脉冲序列,从而创造具有规定的空间和时间可变性的资源景观。在每个化学环境中,可以获得单个尺度的细菌运动动态及其在种群层面的积累,从而能够量化化学战术性能及其对生态相关环境中细菌聚集的影响。

Introduction

微生物依靠化学,检测化学梯度和改变运动的过程,以响应1,导航化学景观,接近营养源和宿主,并逃脱有毒物质。这些微尺度过程决定了微生物与其环境相互作用的宏观动力学2,3。微流体和微加工技术(包括软光刻4)的最新进展彻底改变了我们创造受控微环境的能力,从而研究微生物的相互作用。例如,过去的实验通过产生高可控、稳定的中间至高营养浓度梯度5、6来研究细菌化学。然而,在自然环境中,微尺度化学梯度可能寿命短,通过分子扩散消散,背景条件通常高度稀薄7。为了直接测量首次暴露于不稳定化学环境中的微生物种群的化学反应,我们设计并介绍了将微流体技术与光解相结合的方法,从而模拟野生细菌在自然界中遇到的梯度。

解封技术采用光敏探头,以功能方式将生物分子封装为非活性形式。辐照释放笼状分子,允许生物过程的靶向扰动8。由于细胞化学的快速和精确控制,解开提供9,光解笼化合物传统上使用生物学家,生理学家和神经科学家研究基因10,电通道11和神经元12的激活。最近,科学家利用光分析的显著优势,研究了化学13,确定了暴露于步进化学刺激14、15的单个细菌细胞的旗杆切换动力学,并研究了单精子细胞在三维(3D)梯度16中的运动模式。

在我们的方法中,我们在微流体装置中实现笼体氨基酸的光解,以研究细菌群体对受控化学脉冲的行为反应,这些脉冲通过光释放几乎瞬间可用。使用低放大率(4x)物(NA = 0.13,聚焦深度约40μm)既可观察大视场(3.2 mm x 3.2 mm)上数千种细菌的种群级聚合反应,也可用于单细胞水平的运动测量。我们提出了这种方法的两个应用:1) 释放单个化学脉冲以研究从均匀条件开始的细菌积累 -耗散动力学;2i) 释放多个脉冲来描述在时变、空间异质化学吸引条件下的细菌积累动力学。该方法已在海洋细菌Vibrio ordalii上对氨基酸谷氨酸17进行化学试验,但该方法广泛适用于不同物种和化学吸引剂的组合,以及化学酶以外的生物过程(例如,营养摄入、抗生素暴露、仲裁检测)。这种方法有望帮助阐明真实环境中微生物的生态和行为,并揭示单个细菌在导航瞬时动态梯度时所面临的隐藏权衡。

Protocol

1. 单化学脉冲实验微流体装置的制造 使用计算机辅助设计 (CAD) 软件设计通道并将其打印到透明胶片上以创建照片蒙版(图 1A)。 通过软光刻(在洁净室条件下)制作主人。 用丙酮、甲醇和异丙醇快速连续清洁硅片(4英寸),然后用氮气干燥。在 130°C 的烤箱中烘烤晶圆 5 分钟。 将晶圆放在纺布机的中心,并将 SU-8 光刻胶倒在?…

Representative Results

我们使用微流体和微流体装置(图1)来研究动态营养条件下的细菌积累谱。细菌轨迹是从通过相对比显微镜获得的细菌群体在光解释放的化学脉冲后积累-消散动力学的录影视频中提取的(图2和图3)。通过平均数百万条轨迹,得到了径向漂移速度和细菌浓度的时空动力学。在没有化学梯度的情况下进行游泳的统计数据是在空间和…

Discussion

该方法使研究人员能够在微流体和微流体器件的受控动态梯度下研究细菌化学,从而实现可重现的数据采集。光溶在微尺度上几乎瞬间产生化学脉冲,目的是从各种来源再现细菌在野外遇到的营养脉冲类型,例如,下沉的海洋颗粒25背后的羽状物扩散,或从溶化浮游植物细胞26中传播的营养。

我们提出了这种方法的两个应用:1) 释放单个?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢苏黎世ETH的FIRST微制造设施。这项工作得到了澳大利亚研究理事会发现早期职业研究员奖DE180100911(D.R.B.),戈登和贝蒂摩尔海洋微生物倡议调查员奖GBMF3783(R.S.)和瑞士国家科学基金会赠款的支持。1-002745-000(至 R.S.)。

Materials

(3-Aminopropyl) triethoxysilane (APTES) Sigma-Aldrich A3648 >98% purity, highly toxic
CELLSTAR tube Greiner Bio-One 210261 50 ml
Centrifuge Eppendorf 5424R to eliminate spent media from the bacterial culture
Digital Incubators Incu-Line VWR-CH 390-0384 to bake 3D master
Duster VWR-CH 16650-22 to clean the wafer and microchannels
Hot plate VWR-CH 444-0601 to bond the microchannels
Isopropanol Sigma-Aldrich W292907
LightSafe micro centrifuge tubes Sigma-Aldrich Z688312 1.5 ml
MATLAB Mathworks for image analysis and bacterial tracking
Microcentrifuge tube Eppendorf 30120086 1.5 ml
Microscope glass slide VWR-CH 631-1552
Microscope Nikon Eclipse TiE Nikon Instruments MEA53100 with motorized stage
MNI-Glutamate Tocris Bioscience 1490 >98 % purity, photosensitive
Mold printing equipment Stratasys Objet30 3D printer
Mold printing service 3D Printing Studios Custom https://www.3dprintingstudios.com/
Nanodrop One UV-Vis Spectrophotometer Thermo Fisher Scientific ND-ONE-W to calibrate the uncaging
NIS Elements Nikon Instruments Microscope Imaging Software
Oven Venti-Line VWR-CH 466-3516 to bake PDMS (with forced convection)
Photoresist SU-8-3050 MicroChem Corp. SU8-3050
Plasma chamber Zepto Diener Electronic ZEPTO-1 to functionalize the surfaces before bonding
Polycarbonate membrane Sterlitech PCT0447100 0.4 µm pore size, 19 % open area, 24 µm thickness
Polyethylene microtubing Scientific Commodities BB31695-PE/2 I.D. x O.D.: 0.015" x 0.043" / 0.38mm x 1.09mm
Polystyrene Petri dish VWR-CH 25373-100 bottom surface (90 mm x 15 mm) to bond the millifluidic device
Scale VWR-CH 611-2605 to weight PDMS mixture
sCMOS camera Andor Zyla Oxford Instruments for phase contrast and fluorescence microscopy (max 100 fps)
Sea salt Instant Ocean Product No. SS1-160p
SolidWorks 2015 Dassault Systemes SolidWorks Used to design the mold
Spectra X light engine Lumencolor for LED 395 nm
Sylgard 184 Dow Corning 110-41-155 PDMS Si Elastomer Kit; curing agent
Syringe (Luer-Lok) B Braun Omnifix 4616308F
Syringe Needle Agani A228 from 10 to 30 ml
Syringe Pump 11 Pico Plus Elite Harvard Apparatus 70-4506 Terumo Agani 23 gauge 5/8 inch (16mm)
VeroGrey Stratasys Dual Syringe Pump
Vortex-Genie Scientific Industries SI-0236 Mold Material

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Cite This Article
Carrara, F., Brumley, D. R., Hein, A. M., Yawata, Y., Salek, M. M., Lee, K. S., Sliwerska, E., Levin, S. A., Stocker, R. Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior. J. Vis. Exp. (155), e60589, doi:10.3791/60589 (2020).

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