This work was supported by JSPS KAKENHI grant number JP18K14260 and the budget of Japan Agency for Marine-Earth Science and Technology. We thank Shigeru Deguchi (JAMSTEC) and Tetsuro Ikuta (JAMSTEC) for providing the characterization facilities. We thank Ken Takai (JAMSTEC) for commercial software support. The microfabrication was conducted at Takeda Sentanchi Supercleanroom, The University of Tokyo, supported by &#34;Nanotechnology Platform Program&#34; of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, Grant Number JPMXP09F19UT0087.

The authors have nothing to disclose.

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...

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 microfluidic nanoliter/picoliter/femtoliter reactors1,2,3,4,5,6,7. Analogous to downsizing the feature size of transistors on integrated circuit chips in the semiconductor industry in the past decades, bio/chemical microreactors are going through volume reduction and system integration. Such small-scale tools have had a profound impact on cell-based or cell-free synthetic biology, biomanufacturing, and high-throughput prototyping and screening8,9,10,11,12. This paper describes our recent effort on the development of a unique droplet array technology and demonstrates its application in CFPS13, a fundamental technology for synthetic biology and molecular screening communities14. In particular, we intentionally provide an optimized and low-cost protocol to make the FemDA device accessible to everyone. The low-cost and easy-to-handle protocol for the miniaturized device would contribute to the educational purposes of universities and help spread the technology.
FemDA prepares femtoliter droplets at an ultrahigh density of 106 per 1 cm2 on a planar glass substrate. We coated a hydrophobic polymer, CYTOP15, on the glass substrate and selectively etched (removed) CYTOP at predefined positions to generate a microchamber array on the substrate. Thus, the resulting microchamber is composed of a hydrophobic sidewall (CYTOP) and a hydrophilic bottom (glass). When sequentially flowing water and oil over the patterned surface, the water can be trapped and sealed into the microchambers. The hydrophilic-in-hydrophobic structure is vital for repelling water outside the microchambers, isolating individual microreactors, and retaining a tiny aqueous solution inside the femtoliter space. The unique property was successfully applied for the preparation of water-in-oil droplets and lipid bilayer microcompartments16,17. Compared to the prototype device16, we first optimized the microfabrication process to realize a complete removal of the CYTOP polymer as well as a full exposure of the glass bottom. CYTOP is a special fluoropolymer featuring extremely low surface tension (19 mN/m) lower than that of conventional microreactor materials such as glass, plastics, and silicone. Its good optical, electrical, and chemical performance have already been utilized in surface treatment of microfluidic devices18,19,20,21,22,23,24. In the FemDA system, to achieve good wetting of the oil on the CYTOP surface, the surface tension of the oil must be lower than that of the solid surface25. Otherwise, the liquid oil in contact with the solid surface tends to become spherical rather than spreading over the surface. Besides, we found that some popular perfluorocarbon oils (e.g., 3M FC-40)16 and hydrofluoroether oils (e.g., 3M Novec series) can dissolve CYTOP as a result of the amorphous morphology of CYTOP, which is fatal to quantitative measurement and would be questionable in terms of cross-contamination among droplets. Fortunately, we identified a biocompatible and environmentally friendly oil exhibiting lower (&#60; 19 mN/m) surface tension13. We also found a new surfactant that can dissolve in the selected oil and function in a low concentration (0.1%, at least 10-fold lower than previously reported popular ones26,27)13. The resulting water/oil interface can be stabilized by the surfactant. Because of the high evaporation rate of the oil, following the flush with the oil, we applied another biocompatible and environmentally friendly oil to replace the first one to seal the microchambers. We call the first oil (ASAHIKLIN&#160;AE-3000 with 0.1 wt % SURFLON S-386) the &#8220;flush oil&#8221; and the second oil (Fomblin Y25) the &#8220;sealing oil,&#8221; respectively.
The two-step oil-sealing strategy can realize a robust formation of the femtoliter droplet array within minutes and without sophisticated instrumentation. Due to the evaporation problem, it has been considered challenging to generate microreactors smaller than picoliter volumes28. FemDA addressed this issue by systematically optimizing the materials and processes used for the preparation of microreactors/droplets. Several noteworthy features of the resulting droplets include the high uniformity (or monodispersity), stability, and biocompatibility at the femtoliter scale. The coefficient of variation (CV) of the droplet volume is only 3% (without vignetting correction for the microscopic images), the smallest CV among droplet platforms in the world, which ensures a highly parallel and quantitative measurement. The femtoliter droplet is stable for at least 24 hours without cross-contamination among droplets at room temperature, which is valuable for a reliable time-course measurement. Regarding the biocompatibility, we succeeded in synthesizing various proteins from a single-copy template DNA in the femtoliter droplet, which had previously been considered difficult or inefficient29,30. It would be worthy of elucidating why some proteins capable of being synthesized in the FemDA cannot be synthesized in other droplet systems. FemDA was not merely a technical advancement, but also realized an unprecedentedly quantitative measurement that can correlate the protein yield (as reflected by the fluorescence intensity of the droplet) to the number of template DNA molecules in each droplet. As a result, the histogram of the fluorescence intensity of droplets from FemDA-based CFPS showed a discrete distribution that can be nicely fitted by a sum of Gaussian distributions of equal peak-to-peak intervals. Moreover, the probability of occurrence of droplets containing different numbers of DNA molecules was a perfect fit to a Poisson distribution31. Thus, the different protein yield in each droplet can be normalized based on the discrete distribution. This critical feature allows us to separate the enzymatic activity information from the apparent intensity, that has not been available with other microreactor platforms yet. Existing microfluidic cell/droplet sorting systems are skilled in fully automatic sorting and good at concentrating samples but sometimes can only output a relatively broad or long-tailed histogram in the analytical aspect32,33. Our highly quantitative and biocompatible FemDA system sets a new benchmark and a high analytical standard in the field of microreactor development.
The oils and surfactants that could be used for the preparation of droplets are still very limited34. The combination of ASAHIKLIN AE-3000 and SURFLON S-386 established in FemDA is a new member of the growing arsenal of the physiochemical interface between the aqueous phase and the oil phase13. The new interface in FemDA is physically stable, chemically inert, and biologically compatible with the complex transcription, translation, and post-translational modification machinery for many sorts of proteins13. It would be rather attractive to find a protein that cannot be synthesized in the droplet settings instead. Besides, the cost saving of reagents is more evident in the femtoliter droplet system than that in nanoliter and picoliter reactor systems35,36. In particular, there would often be a large dead volume, which is mainly caused by tubing or external supplies, in microfluidic droplet generation systems but not in our FemDA. The array format is also favored by repeated and detailed microscopic characterization (similar to so-called high-content analysis) for every single reactor37, rather than only a single snapshot for a fast-moving object. The femtoliter scale enabled the integration of over one million reactors on a finger-sized area, while the same number of nanoliter reactors (if it exists) requires over square meter area, which would be undoubtedly impractical to manufacture or use such system.

<table>
	<tbody>
		<tr>
			<td>(3-aminopropyl)triethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Kanto Chemical</td>
			<td>EL grade</td>
			<td>EL: for electronic use.</td>
		</tr>
		<tr>
			<td>3D laser scanning confocal microscope</td>
			<td>Lasertec</td>
			<td>OPTELICS HYBRID</td>
			<td>Other similar microscopes (e.g., Keyence VK-X1000, Olympus LEXT OLS5000) are also applicable.</td>
		</tr>
		<tr>
			<td>50 mL syringe</td>
			<td>Terumo</td>
			<td>SS-50LZ</td>
			<td></td>
		</tr>
		<tr>
			<td>6,8-difluoro-4-methylumbelliferyl phosphate</td>
			<td>Thermo Fisher Scientific</td>
			<td>D6567</td>
			<td>Prepare a 5 mM stock solution in dimethyl sulfoxide</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Kanto Chemical</td>
			<td>EL grade</td>
			<td>EL: for electronic use. 
			Purity 99.8%.</td>
		</tr>
		<tr>
			<td>Air blower</td>
			<td>Hozan</td>
			<td>Z-263</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum block</td>
			<td>BIO-BIK</td>
			<td>AB-24M-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum microtube stand</td>
			<td>BIO-BIK</td>
			<td>AB-136C</td>
			<td></td>
		</tr>
		<tr>
			<td>ASAHIKLIN AE-3000</td>
			<td>AGC</td>
			<td>(Test sample)</td>
			<td>Free test sample may be available upon inquiry to AGC.</td>
		</tr>
		<tr>
			<td>BEMCOT PS-2 wiper</td>
			<td>Ozu</td>
			<td>028208</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch with plunger</td>
			<td>Kai</td>
			<td>BPP-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Matsunami Glass</td>
			<td>No. 1 (24 mm &#215; 32 mm, 0.13~0.17 mm thickness)</td>
			<td>Size-customized.</td>
		</tr>
		<tr>
			<td>Cover glass staining rack</td>
			<td>Nakayama</td>
			<td>803-131-11</td>
			<td></td>
		</tr>
		<tr>
			<td>CRECIA TechnoWipe clean wiper</td>
			<td>Nippon Paper Crecia</td>
			<td>C100-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting mat</td>
			<td>GE Healthcare</td>
			<td>WB100020</td>
			<td></td>
		</tr>
		<tr>
			<td>CYTOP</td>
			<td>AGC</td>
			<td>CTL-816AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Deaeration mixer</td>
			<td>Thinky</td>
			<td>AR-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop cutter</td>
			<td>Roland</td>
			<td>STIKA SV-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 300 MIF</td>
			<td>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.)</td>
		</tr>
		<tr>
			<td>Double-coated adhesive Kapton film tape</td>
			<td>Teraoka Seisakusho</td>
			<td>7602 #25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Kanto Chemical</td>
			<td>EL grade</td>
			<td>EL: for electronic use. 
			Purity 99.5%.</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>Version: ImageJ 1.51n</td>
		</tr>
		<tr>
			<td>Flat-cable cutter</td>
			<td>Tokyo-IDEAL</td>
			<td>MT-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fomblin oil</td>
			<td>Solvay</td>
			<td>Y25, or Y25/6</td>
			<td>Free test sample may be available upon inquiry to Solvay. Fomblin Y25/6 is an alternative if Y25 is not readily available.</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>AS ONE</td>
			<td>TH-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle</td>
			<td>Terumo</td>
			<td>NN-2270C</td>
			<td>22G &#215; 70 mm</td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-E</td>
			<td>Epifluorescence specification, CCD or sCMOS camera, motorized stage, autofocus system, and high NA objective lens are required.</td>
		</tr>
		<tr>
			<td>KaleidaGraph</td>
			<td>Synergy</td>
			<td></td>
			<td>Version: 4.5</td>
		</tr>
		<tr>
			<td>Mask aligner</td>
			<td>SUSS</td>
			<td>MA-6</td>
			<td>Other mask aligners are also applicable as long as the vacuum contact mode is avaliable.</td>
		</tr>
		<tr>
			<td>MICROMAN pipette</td>
			<td>GILSON</td>
			<td>E M250E</td>
			<td>Capillary piston tip: CP250</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Version: 16.16.15</td>
		</tr>
		<tr>
			<td>Mini vacuum chamber</td>
			<td>AS ONE</td>
			<td>MVP-100MV</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>NIPPON GENE</td>
			<td>316-90101</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Amcor</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>NIPPON Genetics</td>
			<td>FG-021D/SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>AS ONE</td>
			<td>GD90-15</td>
			<td>Diameter 90 mm, height 15 mm.</td>
		</tr>
		<tr>
			<td>Photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>AZ P4903</td>
			<td>AZ Electronic Materials was now acquired by Merck. AZ P4620 is an alternative.</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>POWERSCAN HT</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethelene gloves</td>
			<td>AS ONE</td>
			<td>6-896-02</td>
			<td>Trade name: Saniment.</td>
		</tr>
		<tr>
			<td>PURExpress in vitro protein synthesis kit</td>
			<td>New England Biolabs</td>
			<td>E6800S or E6800L</td>
			<td>For cell-free protein synthesis reaction.</td>
		</tr>
		<tr>
			<td>Reactive-ion etching system</td>
			<td>Samco</td>
			<td>RIE-10NR</td>
			<td>Other RIE systems are also applicable but should require optimization of RIE conditions (gas flow rate, chamber pressure, RF power, etching time, etc.)</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>New England Biolabs</td>
			<td>M0314S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotch tape</td>
			<td>3M</td>
			<td>810-1-18D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>194-09575</td>
			<td>8 M concentration; danger.</td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Oshigane</td>
			<td>SC-308</td>
			<td></td>
		</tr>
		<tr>
			<td>SURFLON S-386 surfactant</td>
			<td>AGC</td>
			<td>(Test sample)</td>
			<td>Free test sample may be available upon inquiry to AGC.</td>
		</tr>
		<tr>
			<td>SYLGARD 184 silicone elastomer</td>
			<td>Dow</td>
			<td>Sylgard184</td>
			<td>Chemical composition: polydimethylsiloxane. The default mixing ratio is base : curing agent = 10 : 1 (m/m).</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Ideal-tek</td>
			<td>2WF.SA.1 
			2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>AS ONE</td>
			<td>ASU-2M</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum chuck</td>
			<td>Oshigane</td>
			<td>(Customized)</td>
			<td>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 &#215; 32 mm).</td>
		</tr>
	</tbody>
</table>

a femtoliter droplet array for massively parallel protein synthesis from single dna molecules

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.

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...

Watch this Scientific Journal Video about A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules at JoVE.com

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

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.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

a-femtoliter-droplet-array-for-massively-parallel-protein-synthesis

Research

JoVE Journal

Chemistry

10.2K Views.  Japan Agency for Marine-Earth Science and Technology (JAMSTEC). 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.

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

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction

This protocol presents both the synthesis method of the Ni single atom catalyst, and the electrochemical testing of its catalytic activity and selectivity in aqueous CO2 reduction. Different from traditional metal nanocrystals, the synthesis of metal single atoms involves a matrix material that can confine those single atoms and prevent them from aggregation. We report an electrospinning and thermal annealing method to prepare Ni single atoms dispersed and coordinated in a graphene shell, as active centers for CO2 reduction to CO. During the synthesis, N dopants play a critical role in generating graphene vacancies to trap Ni atoms. Aberration-corrected scanning transmission electron microscopy and three-dimensional atom probe tomography were employed to identify the single Ni atomic sites in graphene vacancies. Detailed setup of electrochemical CO2 reduction apparatus coupled with an on-line gas chromatography is also demonstrated. Compared to metallic Ni, Ni single atom catalyst exhibit dramatically improved CO2 reduction and suppressed H2 evolution side reaction.

Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction

Here, we present a protocol for the synthesis and electrochemical testing of transition metal single atoms coordinated in graphene vacancies as active centers for selective carbon dioxide reduction to carbon monoxide in aqueous solutions.

This protocol presents both the synthesis method of the Ni single atom catalyst, and the electrochemical testing of its catalytic activity and selectivity ...

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...