A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Summary

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm² planar substrate that can be used for cell-free protein synthesis.

Abstract

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

Introduction

Researchers use reactors to carry out bio/chemical reactions. There are significant efforts that have been made to reduce the size of the reactor and increase the experimental throughput in order to lower the reagent consumption while improving the work efficiency. Both aspects aim to liberate researchers from a heavy workload, decreasing the cost and speeding up research and development. We have a clear historical roadmap about the development of the reactor technologies from the viewpoint of reaction volumes and throughput: single beakers/flask/test-tubes, milliliter tubes, microliter tubes, microliter 8-tube strips, microliter 96/384/1536-well plate, and microfluid....

Protocol

1. Microfabrication of the femtoliter microchamber array substrate

NOTE: Conduct the following microfabrication experiment in a cleanroom. Wear gloves and a cleanroom suit before entering the cleanroom.

1. Cleaning cover glass substrate
   1. Set the cover glass on a cover glass staining rack. Sonicate the cover glass in 8 M sodium hydroxide (NaOH) for 15 min at room temperature (RT).
      CAUTION: NaOH in high concentrations is highly dangerous to skin and eye. Gently handle it without any splash.
   2. Take the rack out of the NaOH solution and rinse the cover glass using water for ten times. Keep the cover glass in....

Results

The microfabrication process consists of substrate cleaning, surface functionalization, CYTOP coating, photolithography, dry etching, photoresist stripping, and final cleaning. Importantly, the presented protocol allowed complete removal of the hydrophobic CYTOP polymer inside the microchambers Figure 3A), producing a highly parallel hydrophilic-in-hydrophobic structure on a standard cover glass substrate. With the aid of the oil sealing protocol, the uniform dimension of the resulting dropl.......

Discussion

The highly quantitative measurement based on the highly uniform, stable, and biocompatible droplets in FemDA enabled the discrete distribution, the unique feature of our study differing from others. We systematically optimized and detailed the microfabrication and droplet formation processes in this paper. There are several critical steps in the established protocol.

First, the uniform coating of highly viscous CYTOP polymer on the rectangular thin glass substrate largely determines the qualit.......

Materials

Name	Company	Catalog Number	Comments
(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).