This research work is supported as part of the Center for Atomic Level Catalyst Design, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001058 and also supported by Board of Regents under grants award number LEQSF (2009-14)-EFRC-MATCH and LEDSF-EPS(2012)-OPT-IN-15. MRCAT operations are supported by the Department of Energy and the MRCAT member institutions. The use of the Advanced Photon Source at ANL is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Financial support for JTM was provided as part of the Institute for Atom-efficient Chemical Transformations (IACT), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences.

Millifluidics set-up: Purchase a millifluidic chip (made of polyester terephthalate polymer) from Microplumbers Microsciences LLC, which has serpentine channels with dimensions of 2 mm (W) x 0.15 mm (H) x 220 mm (L). Use FEP Tubing &#160;with dimensions of 0.25 mm I.D., 1/16 in&#160;O.D., for connecting the chip to the pump. Use two different pumps for the two different experiments. Use P-Pump&#160;for the first experiment (copper nanoparticles) and the millifluidic device&#160;for the second experiment (gold nanoparticles). To minimize the problem of gas bubbles within the channels, freshly prepared NaBH4 solution was left open to stand for ~10-15 min before pumping into the chip so that the gas bubbles escape from the solution. This step was followed for all of our experiments.
1. Synthesis of Ultra-small Copper Nano Clusters (UCNCs)
<ol>
	<li>Chemicals required: Obtain copper(II) nitrate hydrate, sodium borohydride, sodium hydroxide pellets and O-[2-(3-mercaptopropionylamino)ethyl]-O&#39;-methylpolyethylene glycol (MW=5,000) [MPEG] and use all chemicals without further purification. Use nanopure water (18.2 M&#937;-cm) for the experiment.</li>
	<li>Use P-Pump regulated under nitrogen pressure for the experiment. Test the pumps with water as solvent at different pressures prior to the experiment to correlate with the corresponding flow-rates (ml/hr). Rinse the millifluidic reactor and tubing with deionized water before initiation of the experiment.</li>
	<li>Dissolve 174 mg (0.95 mmol) of copper(II) nitrate and 610 mg (0.122 mmol) of O-[2-(3-mercaptopropionylamino)ethyl]-O&#39;-methylpolyethylene glycol in 28 ml of nanopure water and keep them in a vial to be connected with one input channel</li>
	<li>Keep another solution of 111 mg (2.93 mmol) of sodium borohydride and 102 mg (2.78 mmol) sodium hydroxide in 28 ml (pH ~13) in a different vial and connect it with the other input channel.</li>
	<li>Flow both the solutions simultaneously within the millifluidic reactor at different flow-rates (given below) and collect the resulting UCNCs at the outlet in glass vial. Purge the solution with nitrogen and store it under nitrogen.</li>
	<li>Operate the pumps under different constant pressures of 50 mbar (6.81 ml/hr), 100 mbar (14.31 ml/hr), 200 mbar (32.7 ml/hr) and 300 mbar (51.4 ml/hr) at room temperature for the synthesis of UCNCs at different flow-rates.</li>
</ol>
While the synthesis procedure was demonstrated using the millifluidic set-up with P-Pump, it can also be carried out using the hand-held millifluidic device from Millifluidica.
2. Time Resolved In situ Kinetic Studies on Gold Nanoparticle Formation
<ol>
	<li>Chemicals required: Obtain chloroauric acid (HAuCl4.3H2O) meso-2,3-dimercaptosuccinic acid (DMSA) and sodium borohydride&#160;and use all chemicals without further purification. Use nanopure water (18.2 M&#937;-cm) for the experiment.</li>
	<li>Use high precision, fully automated, pulsation free syringe pumps&#160;to flow the liquids within the chip. Test the pumps with water as solvent at different flow-rates prior to the experiment to optimize the required flow-rate.</li>
	<li>Prepare standard solutions of (i) HAuCl4.3H2O (10 mmol, 118.2 mg/30 ml) and (ii) DMSA (20 mmol, 109.2 mg/30 ml) with 50 mg of sodium hydroxide (pH 12) in nanopure water.</li>
	<li>Feed the two solutions through two separate syringes into the millifluidic chip at a constant flow-rate of 10 ml/hr using the automated pump.</li>
	<li>Couple the millifluidic chip to the synchrotron beam line using a metal stage that has access to movement in XYZ directions and collect the XAS data at different zones on the chip as the solutions were pumped through the chip.</li>
</ol>
While the in situ analysis procedure was demonstrated using the millifluidic set-up with P-Pump, it can also be carried out using a hand-held millifluidic device.
3. Continuous Flow Gold Catalysis
This procedure was demonstrated using a hand-held millifluidic device.
<ol>
	<li>Chemicals required: Obtain chloroauric acid (HAuCl4.3H2O), meso-2,3-dimercaptosuccinic acid (DMSA), sodium borohydride, 4-nitrophenol, 4-aminophenol&#160;and use all the chemicals without further purification. Use nanopure water (18.2 M&#937;-cm) for the experiment.</li>
	<li>Catalyst preparation: Prepare standard solutions of HAuCl4.3H2O (10 mmol, 118.2 mg/30 ml), DMSA (20 mmol, 109.2 mg/30 ml) and NaBH4 (10 mmol, 11.34 mg/30 ml) in Nanopure water.</li>
	<li>Take 10 ml each of HAuCl4 and DMSA solutions into two separate vials and flow them within the chip using the hand-held millifluidic device with a uniform flow-rate of 12 ml/hr for 45 min.</li>
	<li>Flow 10 mmol NaBH4 within the chip at 12 ml/hr flow-rate for 15 min to reduce the Au(I) to Au(0).</li>
	<li>Finally, wash the chip with nanopure water for 30 min at the same flow-rate before conducting the catalysis experiments.</li>
	<li>Catalysis reaction: Perform the chemical conversion reaction (reduction) of 4-nitrophenol (4-NP) into 4-aminophenol (4-AP) within the gold catalyst (prepared above) coated millifluidic channel as given below.</li>
	<li>Mix 15 ml of 9 x&#160;10-5 mol solution of 4-NP with 3.3 ml of 0.65 mol NaBH4 solution to form 4-nitrophenolate ion (4-NPI).</li>
	<li>Pass the resultant solution over the gold catalyst deposited within chip at a constant flow-rate of 5 ml/hr to evaluate the catalytic activity. Analyze the UV-Vis spectra&#160;of the collected products within the wavelength range of 250-500 nm to confirm the conversion of 4-NP.</li>
	<li>Estimate the catalytic activity of the reaction by obtaining the calibration curve of 4-NPI. Calibration curve can be acquired by plotting the experimentally observed absorption intensity (I) of 4-NPI at different standard concentrations. The peak heights (at 399 nm) for the UV-Vis absorption curves represent the absorption intensity (I) values and according to the Beer Lambert&#39;s law, any change in the peak height value would show corresponding change in its concentration. Therefore, estimate the catalytic activity by finding the difference in initial and final concentrations of the reactant from the calibration curve. For example, if the peak height is 1 unit (Figure 6) it corresponds to a catalytic conversion of 90% (based on the calibration plot).</li>
</ol>

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All authors except C.S.S.R. Kumar declare that they have no competing financial interests. C .S.S.R. Kumar is the founder of the company Millifluidica LLC.

The UCNCs were formed by the reduction reaction of copper nitrate with sodium borohydride in the presence of the polymeric capping agent O-[2-(3-Mercaptopropionylamino)ethyl]-O&#39;-methylpolyethylene glycol (MW=5,000) [MPEG]. The reaction was performed within the millifluidic chip reactor at different flow-rates such as 6.8 ml/hr, 14.3 ml/hr, 32.7 ml/hr, and 51.4 ml/hr to study the effect of flow-rates on the UCNCs formed. The respective residence times for the above flow-rates are 47.49, 24.44, 16.56, and 9.02 sec. The...

Lab-on-a-chip (LOC) devices for chemical synthesis have demonstrated significant advantage in terms of increased mass and heat transfer, superior reaction control, high throughput and safer operation environment1. These devices can be broadly classified into chip based fluidics and nonchip based fluidic devices. Among the chip-based fluidics, microfluidics is well investigated and a topic well-covered in the literature2-5. Nonchip based LOC systems use tubular reactors6. Conventionally, microfluidic systems are used for precise control and manipulation of fluids that are geometrically constrained to submillimeter scale. We have recently introduced the concept of chip-based millifluidics, which can be used for manipulation of fluids in channels in millimeter scale (either width or depth or both of the channels are at least a millimeter in size)7-9. Furthermore, the millifluidic chips are relatively easy to fabricate while offering similar control over flow-rates and manipulation of reagents. These chips could also be operated at higher flow-rates, creating smaller residence times, thereby, offering the possibility for scale-up of controlled synthesis of nanoparticles with narrower size distribution. As an example, we have recently demonstrated the synthesis of ultra-small copper nanoclusters and characterized them using in situ X-ray absorption spectroscopy as well as TEM. Ability to obtain small residence times within millifluidic channels in combination with the use of MPEG, which is very efficient bidentate PEGylated stabilizing agent for the formation of stable colloids of copper nanoclusters7 .
In addition to the synthesis of chemicals and nanomaterials, the millifluidics could offer, due to higher volume and concentration at the probe area, a synthetic platform that is more generalized and efficient for time-resolved kinetic studies and also achieves better signal to noise ratio than microfluidic systems7,10. We show the use of millifluidic chip as an example for time resolved analysis of the growth of gold nanostructures from solution using in situ XAS with a time resolution as small as 5 msec11.
Also, majority of the micro reactors developed to date for catalysis applications are based on silicon12,13. Their expensive fabrication in addition to small volumes generated makes them unsuitable for large scale manufacturing. The two general methods for coating the channels with nanocatalysts - chemical and physical, often referred to as silicon coating procedures, are currently in vogue14,15 . In addition to expensive micro fabrication, clogging of the channels makes micro reactor catalysis may be unsuitable for large-scale manufacturing. Although microreactors have been used for heterogeneous catalysis in micro continuous flow-through processes earlier16-18 , the ability to control the dimension, and morphology of the embedded gold nanostructured catalysts within continuous flow channels was never explored before. We have recently developed a technology for coating the millifluidic channels with Au catalysts, having controlled nano morphology and dimensions (Figure 5)11, for carrying out catalysis of industrially important chemical reactions. As an example we have demonstrated conversion of 4-nitrophenol into 4-aminophenol catalyzed by nanostructured gold coated within the millifluidic channels. Considering that a single millifluidic reactor chip can produce flow-rates of 50-60 ml/hr,7 high-throughput and controlled synthesis of chemicals is possible either through continuous flow operation or parallel processing.
In order to capitalize on the possibilities the millifluidics offer, with few examples described as above, we also demonstrate a user-friendly millifluidic device that is portable and has the all the required components such as millifluidic chips, manifolds, flow controllers, pumps and electrical connections integrated. Such a millifluidic device, as shown in the Figure 7, is now available from the company Millifluidica LLC (<a href="http://www.millifluidica.com" target="_blank">www.millifluidica.com</a>). The manuscript also provides protocols using the hand-held millifluidic device, as described below, for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

<table><tbody><tr><td>Copper (II) nitrate hydrate</td><td>Sigma-Aldrich</td><td>13778-31-9</td><td>99.999% pure</td></tr><tr><td>O-[2-(3-mercaptopropionylamino)ethyl]-O&amp;prime;-methylpolyethylene glycol</td><td>Sigma-Aldrich</td><td>401916-61-8</td><td>MW=5,000</td></tr><tr><td>HAuCl&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;.&lt;/sup&gt;3H&lt;sub&gt;2&lt;/sub&gt;O (Chloroauric acid)</td><td>Sigma-Aldrich</td><td>27988-77-8</td><td>99.999% pure</td></tr><tr><td>meso-2,3-dimercaptosuccinic acid (DMSA)</td><td>Sigma-Aldrich</td><td>304-55-2</td><td>~98% pure</td></tr><tr><td>4-Nitrophenol</td><td>Sigma-Aldrich</td><td>100-02-7</td><td>spectrophotometric grade</td></tr><tr><td>4-Aminophenol</td><td>Sigma-Aldrich</td><td>123-30-8</td><td>&gt;99% pure (HPLC grade)</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>16940-66-2</td><td>98% pure</td></tr><tr><td>Sodium hydroxide pellets</td><td>Sigma-Aldrich</td><td>1310-73-2</td><td>99.99% pure</td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Millifluidic Chips</td><td>Microplumbers Microsciences LLC</td><td>SDC-01</td><td>Made from polyester terephthalate polymer</td></tr><tr><td>Pressure Pump</td><td>Mitos P-Pump, Dolomite</td><td>3200016</td><td></td></tr><tr><td>Automated Syringe Pump</td><td>Cetoni Automation and Microsystems, GmbH</td><td></td><td>Syringe pump neMESYS</td></tr><tr><td>UV-3600 UV-VIS-NIR Spectrophotometer</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>Hand-held Millifluidic Device</td><td>Millifluidica</td><td>SCMD-1008</td><td>&lt;strong&gt;Figure 7&lt;/strong&gt;</td></tr></tbody></table>

millifluidics for chemical synthesis and time-resolved mechanistic studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Well dispersed and uniform sized copper nanoclusters with a narrow size distribution were obtained using the millifluidic chip setup (Fig. 1a). The different flow-rates used for synthesis did not have a significant effect on the size of the clusters. Nevertheless, with increase in the flow-rate, there is an observable improvement in the narrowing of the size distribution. UCNCs with a best narrow size distribution were obtained at a flow-rate of 32.7 mL/hr. The size of UCNCs formed at 32.7 mL/hr flow-rate has an average ...

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Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

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11.1K Views.  Louisiana State University. Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

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Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Chemistry

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

Engineering

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 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 ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

Biochemistry

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

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Fluorescence detection methods for microfluidic droplet platforms

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

Microfluidic Mixers for Studying Protein Folding

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

A Microfluidic Chip for ICPMS Sample Introduction

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

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

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device