Authors would like to thank the financial support from Swiss National Science Foundation (SNF) through the project no. 200021_160174.

Note: Two layers of a double-layer microfluidic device are designed using a drawing software, e.g., AutoCAD and printed to form high-resolution film masks, with a feature precision limit of 5 &#181;m. Master molds are created by SU-8 lithography on 4&#34; silicon wafers, allowing the production of structures 50 &#181;m in height.
1. Master Mold Fabrication Using SU-8 Photolithography
<ol>
	<li>Place the silicon wafer on a hot plate set at 200 &#176;C for 10 min to dehydrate. 
	Note: Silicon wafer dehydration provides a better contact and ensures the spreading of the SU-8 photoresist during the spin coating step.</li>
	<li>Cool the dehydrated wafer down to room temperature over a period of 3 min.</li>
	<li>Load the wafer onto a spin coater and deposit 4 ml of SU-8 photoresist (approximately 1 ml of SU-8 per inch of substrate) at the center of the wafer.
	<ol>
		<li>First, spread the deposited SU-8 slowly at 500 revolutions per minute (rpm) for 10 sec. Such a rotational speed ensures that SU-8 coverage is increased over the entire wafer surface.</li>
		<li>Second, control the thickness of the SU-8 by spinning the substrate at higher speeds. In the current experiments, use a spin speed of 3,000 rpm for 30 sec to generate SU-8 features 50 &#181;m high.</li>
	</ol>
	</li>
	<li>Wipe the edge bead of the wafer carefully with a cotton wipe while spinning at a low rpm (typically 100 rpm).</li>
	<li>Heat the spin-coated wafer on a hot plate at 95 &#176;C for 15 min to remove residual solvent from the SU-8 (i.e., &#34;soft bake&#34;). 
	Note: The presence of patterns or &#34;wrinkles&#34; in the resist layer indicates the incomplete removal of solvent.</li>
	<li>Cool the baked wafer back down to room temperature and contact the emulsion printed side of the photomask with the wafer prior to exposure.</li>
	<li>Turn on the UV-lamp and exposure unit and let the system stabilize over a period of 10 min. Measure the lamp intensity at 365 nm using an UV-optometer, and estimate the required exposure time (according to time = exposure energy/intensity at 365 nm). 
	Note: The exposure energy in the current experiments was calculated to be 250 mJ/cm2.</li>
	<li>Expose the photomask on the spin-coated wafer to UV light for the time estimated in the previous step. Here, expose for 79.6 sec.</li>
	<li>Immediately after exposure, bake the exposed wafer on a hot plate at 65 &#176;C for 1 min and subsequently at 95 &#176;C for 5 min. In this step, the reaction is initiated by UV -light and completed after baking.</li>
	<li>Leave the post-baked wafer to cool down to room temperature over a period of 3 min.</li>
	<li>Develop the non-crosslinked SU-8 on the wafer by dissolving it in the SU-8 developer over 8 min. To ensure complete removal of non-crosslinked SU-8, split the process into two steps.
	<ol>
		<li>In the first, immerse the wafer in the developer solution for 5 min, removing the majority of non-crosslinked SU-8.</li>
		<li>Then immerse the wafer in a fresh solution of developer for 3 min to dissolve the remaining non-crosslinked SU-8 (typically trapped between crosslinked structures).</li>
	</ol>
	</li>
	<li>Rinse the developed wafer with isopropanol and let the wafer having patterned structures (hereinafter referred to as &#34;master mold&#34;) stand to dry out. Observation of a milky residue upon rinsing the master mold indicates that development is incomplete.</li>
	<li>Heat the dried master mold on a hot plate at 200 &#176;C for 2-5 min to &#34;hard bake&#34; the substrate and anneal potential cracks in the resist.</li>
	<li>Allow the fabricated master mold to cool down to room temperature.</li>
	<li>Place the master mold in a desiccator (connected to a vacuum pump) inside a fume hood.</li>
	<li>Pour 100 &#181;l of trimethylsilyl chloride (TMCS) into a glass beaker and place this inside the desiccator. 
	CAUTION: TMCS is flammable, corrosive and toxic; thus, handling steps should be performed under a fume hood, with the user wearing protective gloves, goggles and a lab coat.</li>
	<li>Put the desiccator under vacuum and wait at least 1 hr to allow the TMCS vapor to deposit onto the master mold surface.</li>
	<li>After 1 hr, slowly equilibrate the pressure within desiccator and open to the atmosphere. 
	CAUTION: Do not breathe directly above the open desiccator.</li>
	<li>Remove the silanized master and close the desiccator.</li>
</ol>
2. Fabrication of Double-layer Microfluidic Devices
Note: The protocol is particularly sensitive to the time and temperature. Any failure to follow to the time frame and temperature may lead to fabrication of non-bonded, and therefore, non-functional devices.
<ol>
	<li>Pour a mixture of PDMS elastomer and curing agent (5:1 in weight) into a disposable weighing dish and completely mix with a plastic spatula. In the current experiments, use 50 g of elastomer and 10 g of curing agent to form a PDMS layer approximately 5 mm in height19,26.</li>
	<li>Place the well mixed PDMS in a desiccator under vacuum to degas and remove trapped bubbles for 15 min.</li>
	<li>Mix PDMS elastomer and curing agent (20:1 in weight) in a disposable weighing dish (e.g., 10 g elastomer and 0.5 g curing agent)19,26.</li>
	<li>Fix the &#34;control layer&#34; master mold in a frame (in the current experiments, an 11 mm round polytetrafluoroethylene (PTFE) ring).</li>
	<li>After 15 min, place the 20:1 PDMS mixture in the desiccator under vacuum for degassing.</li>
	<li>At the same time as the previous step, take out the 5:1 PDMS mixture from the desiccator and pour this onto the &#34;control layer&#34; master mold that is located inside the round frame. Place the frame containing the PDMS and master mold into the desiccator under vacuum as well. Keep the surplus PDMS.</li>
	<li>After 45 min (and 30 min after putting both PDMS mixtures into the desiccator), take both PDMS mixtures out of the desiccator and place the frame containing 5:1 PDMS and the &#34;control layer&#34; master mold in an oven at 80 &#176;C.</li>
	<li>At the same time, begin to spin coat the &#34;fluidic layer&#34; master mold with the 20:1 PDMS mixture. The rotational speed for spin coating is determined based on the desired height, and has been reported elsewhere27. Aim to terminate spin coating at 60 min and keep the residual PDMS.</li>
	<li>After 60 min, put the &#34;fluidic layer&#34; master mold spin coated with 20:1 PDMS into an oven at 80 &#176;C.</li>
	<li>At 75 min, take both master molds out of the oven.</li>
	<li>Peel off only the 5:1 PDMS from the &#34;control layer&#34; master mold, dice the substrate with a blade and punch the holes for control layers with a 1 mm biopsy puncher at the inlets positions determined in the design. Here, the control layer is 24 mm in length and 24 mm in width.</li>
	<li>Remove debris from diced chips using adhesive tape.</li>
	<li>Manually assemble the diced and punched control layer chips on top of the 20:1 PDMS spin coated on the &#34;fluidic layer&#34; master mold using a stereomicroscope with the magnification of 500X (Figure 1).</li>
	<li>Pour and draw the residual PDMS around the assembled chips to make a thicker PDMS layer and thereby facilitate removal of the bonded fluidic and control layers at the end.</li>
	<li>Place the &#34;fluidic layer&#34; master mold containing the two layer devices in an oven at 80 &#176;C and store overnight.</li>
	<li>The following day, take the cured assembly out of the oven and allow it to cool down to room temperature.</li>
	<li>Peel off the PDMS assembly from the &#34;fluidic layer&#34; master mold, dice the fabricated double-layer devices with a blade (24 mm in length and 24 mm in width) and punch fluidic inlets/outlets with a 1.5 mm biopsy puncher.</li>
	<li>Treat the surface of chips with open channels and glass coverslips (24 mm &#215; 40 mm) with a corona discharge and immediately bond them together. Treat by moving the corona discharge over the PDMS slab and glass coverslip over 1 min. Alternatively, use a benchtop plasma system to facilitate bonding.</li>
	<li>Store the bonded double-layer chips in an oven at 70-80 &#176;C for at least 4 hr.</li>
</ol>
3. Microfluidic System Assembly
<ol>
	<li>After the microfluidic device has been assembled, connect the fluidic layer inlets of the chip to the fluidic reservoirs (syringes) using polytetrafluoroethylene (PTFE) tubing (0.8 mm id).</li>
	<li>Connect the pressure supply source to the control layer inlets using silicone rubber tubing and metal connectors that have an external diameter of 1.6 mm.</li>
	<li>Open and close the valves by applying pressurized air at 3 bar using a pressure source which is operated manually. Supply fluids to the channels using a series of computer controlled syringe pumps.</li>
	<li>Visualize actuation of valves and device operation with a high resolution camera mounted on an inverted microscope. Use 5X to 63X magnification.</li>
</ol>
4. Manipulation of the Laminar Flow Regime by Pneumatic Cage Actuation
Note: The fluidic layer consists of two inlet converging channels, which are 150 &#181;m in width, to a wider main channel 300 &#181;m in width. And the control layer has a series of identical rectangular valves (250 &#181;m &#215; 200 &#181;m) that are located on top of the main fluidic channel.
<ol>
	<li>Once the set-up is connected to the syringe pump and pneumatic controller systems, introduce an aqueous dye flow via one of the inlet channels at the flow rate of 20 &#181;l/min.</li>
	<li>Close the valve by actuating it at 3 bar.</li>
	<li>Be aware that the fluid can still flow around the valve. This feature is important in achieving controlled chemical treatment of trapped structures18,25.</li>
	<li>Open the valve by releasing the pressure.</li>
	<li>While the dye solution flows through the first channel, inject another aqueous fluid into the second inlet channel at 20 &#181;l/min. An interface between two aqueous flows is formed due to the laminar flow regime present in the microfluidic device.</li>
	<li>Close the valve by actuating it at 3 bar. In this case, the actuation of the valve changes the interface of the two aqueous flows; a result that can be used to modify the synthetic pathway during the formation of a CP (vide infra)18,28.</li>
	<li>Change the fluid flow rates to 30 &#181;l/min and 10 &#181;l/min and observe the down- or up-shifted guiding of the interface generated between the two fluids.</li>
</ol>
5. Localization of Microparticles
<ol>
	<li>Connect the fabricated double-layer chip to the syringe pump and pneumatic controller systems.</li>
	<li>Prepare an aqueous solution containing 10 wt.% polystyrene fluorescent particles (5 &#181;m in diameter, excitation and emission max at 468 nm and 508 nm, respectively).</li>
	<li>Use laser excitation source operating at a wavelength of 488 nm.</li>
	<li>Introduce the particle-laden fluid into the two inlet channels at a total flow rate of 20 &#181;l/min.</li>
	<li>Wait for 2 min until a stable flow is established.</li>
	<li>Actuate the valve at 3 bar to close it. Several particles will be trapped underneath the valve and localized on the surface whilst the flow is maintained.</li>
</ol>
6. Generation and Controlled Reduction of a Coordination Polymer (CP)
<ol>
	<li>Prepare a 2.5 mM aqueous solution of silver nitrate (AgNO3).</li>
	<li>Prepare a 2.5 mM aqueous solution of cysteine (Cys).</li>
	<li>Prepare a saturated ascorbic acid solution in ethanol18.</li>
	<li>Use the same double-layer chip and inject the two reagents into the two inlet channels (one reagent per inlet) each at a flow rate of 50 &#181;l/min.</li>
	<li>Observe the formation of silver and cysteine (Ag(I)Cys) CPs at the interface of two co-flowing steams.</li>
	<li>Actuate the valve at 3 bar to trap the formed Ag(I)Cys CPs underneath the valve.</li>
	<li>Flush distilled water into the inlet channels at a flow rate of 50 &#181;l/min to wash away the surplus reagents used during the synthetic process.</li>
	<li>Release the pressure and open the valve. The generated CPs remain underneath the valve under stopped-flow conditions.</li>
	<li>In order to conduct a controlled chemical reduction of trapped Ag(I)Cys CPs, release the valve pressure at 1 bar and flush the saturated ascorbic acid solution in ethanol at a flow rate of 10 &#181;l/min.</li>
	<li>Observe a clear color change that is attributed to the reduction of Ag(I) to metallic silver (Ag(0)) by the ascorbic acid18.</li>
</ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nicolet iS5 User Guide. , (2015).</li></ol>

The authors have nothing to disclose.

The reported approach can be easily modified to fabricate different valve shapes to afford other applications such as fluid confinement. Indeed, the flexibility of this protocol also allows for modification of the thickness of the bottom layer, and thereby of the PDMS membrane, from a couple of tens to a few hundreds of microns to fulfill any application of interest. Moreover, dimensions of structures in each layer of the device can be optimized for the desired application and various heights of structures on the master molds can be simply achieved by spinning the photoresist at different velocities. Spinning the photoresist at a higher speed results in thinner structures. 
To better implement the protocol, a clean room environment for the fabrication of the master molds is substantially essential; otherwise, the fabrication procedure will lead to defective master molds and thereby to unusable microfluidic devices. Two critical aspects should be emphasized in this protocol: i) the constant temperature of the oven that needs to be adjusted to 80 &#176;C and ii) the programmed time period between processes that has to be complied accurately. Any modification of temperature and time frame in the protocol might lead to non-bonded chips, and thus, to non-functional devices.
The "turbulent free" conditions typically encountered in microfluidic systems have recently been employed for the generation of microstructures or molecular materials inside30 and outside single layer microfluidic chips31. In double-layer microfluidic chips, the laminar flow regime, and hence, the interface generated between continuous co-flows can be manipulated using pneumatic cages18,28. These devices also provide for effective control over the synthetic pathway, which in turn leads to precise localization and trapping on surfaces18. 
As mentioned earlier, pneumatic actuation in double-layer microfluidic chips has been previously employed for various applications such as cell trapping20, enzymatic activity studies21 and protein crystallization24. However, the main objective of the reported approach is to propose a platform to be used for trapping and directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures18,25. 
The described method does not only allow trapping of anisotropic structures but can be used to localize particles onto surfaces. Future studies can be effectively directed towards the design of new valve shapes for additional application in biology, materials science and sensor technologies. The combination of different valve shapes as well as altered channel heights and membrane thicknesses can be employed to fulfill specific applications, such as chemical studies based on diffusional mixing and the localization of material growth.
A further application of the described microfluidic platforms is in the controlled chemical doping of crystals, which can lead to a rationalized formation of interfaces in crystalline structures19. This approach also provides for a wide range of post-treatments of on-chip trapped structures; a methodology that will undoubtedly open new horizons in materials engineering.
It is important to underline that the number of technologies enabling controlled chemical reactions under dynamic conditions and onto crystalline matter are very limited at present, hence making this approach very attractive in materials-related fields. However, a major limitation of this technology is the use of PDMS. PDMS elastomer is incompatible with many organic solvents, which limits the number of reactions that can be conducted inside these microfluidic chips. In future, the development of other elastomers that can tolerate and be stable against a broader number of organic solvents will be highly required in order to expand this field of research to other materials and chemistries.

Molecular materials have long been studied in the scientific community because of their broad number of applications in fields such as molecular electronics, optics and sensors1-4. Amongst these, organic conductors are an especially exciting class of molecular materials because of their central role in flexible displays and integrated functional devices5,6. However, methodologies used to enable electronic charge transport in molecular-based materials are restricted to the formation of charge transport complexes (CTCs) and charge transport salts (CTSs)7-10. Frequently, CTCs and CTSs are generated by electrochemical methods or by direct chemical redox reactions; processes that hamper a controlled transformation of donor or acceptor moieties to more complex architectures where multifunctionality can be conceived. Accordingly, the elucidation of new systematic methods for the controllable generation and manipulation of molecular-based materials remains a significant challenge in the fields of materials science and molecular engineering, and if successful will undoubtedly lead to new functions and novel technological applications.
In this context, microfluidic technologies have recently been used to synthesize molecular-based materials due to their ability to control heat and mass transfer as well as the reaction-diffusion volume of reagents during a synthetic process11,12. Put simply, in continuous flows and at low Reynolds numbers a stable interface between two or more reagent streams can be achieved, which affords the formation of a well-controlled reaction zone inside the flow path, where mixing only occurs through diffusion13-16. Indeed, we have previously employed laminar flows to localize the synthetic pathway of crystalline molecular materials such as coordination polymers (CPs) inside microfluidic channels17. Although this methodology has shown great promise in realizing novel CP nanostructures, the direct integration of such structures onto surfaces, as well as controlled chemical treatment after their formation has yet to be realized in situ18. To overcome this limitation, we have recently shown that the actuation of microfluidic pneumatic cages (or valves) incorporated in two-layer microfluidic devices can advantageously be used in this respect. Since the pioneering work of Quake&#39;s group19, microfluidic pneumatic valves have frequently been used for single-cell trapping and isolation20, enzymatic activity investigations21, trapping of small fluid volumes22, localization of functional materials on surfaces23 and protein crystallization24. However, we have shown that double layer microfluidic devices can be used to trap, localize and integrate in situ formed structures to read out components and on surfaces18. Furthermore, we have also demonstrated that such technology can be used to perform controlled chemical treatments on trapped structures, enabling both, &#34;microfluidic assisted ligand exchange&#34;18 and controlled chemical doping of organic crystals18,25. In both cases, CTCs could be synthesized under controlled microfluidic conditions, and in the most recent study, multifunctionality could be described in the same material piece. Herein, we demonstrate the performance of these double-layer microfluidic devices employing dye-laden flows, generate and control the coordination pathway of a CP as well as its localization on the surface of a microfluidic channel and finally assess controlled chemical treatments onto on-chip trapped structures.

<table><tbody><tr><td>High resolution film masks</td><td>Microlitho, UK</td><td>-</td><td>Features down to 5 &amp;#956;m</td></tr><tr><td>SU8 photoresist</td><td>MicroChem Corp., USA</td><td>SU8-3050</td><td>-</td></tr><tr><td>Silicon wafers</td><td>Silicon Materials Inc., Germany</td><td>4&quot; Silicon Wafers</td><td>Front surface: polished, Back surface: etched</td></tr><tr><td>Silicone Elastomer KIT (PDMS)</td><td>Dow Corning, USA</td><td>Sylgard&amp;reg; 184</td><td>-</td></tr><tr><td>Spinner</td><td>Suiss MicroTech, Germany</td><td>Delta 80 spinner</td><td>-</td></tr><tr><td>UV-Optometer</td><td>Gigahertz-Optik Inc., USA</td><td>X1-1</td><td>-</td></tr><tr><td>Mask Aligner</td><td>Suiss MicroTech, Germany</td><td>Karl Suss MA/BA6&amp;nbsp;</td><td>-</td></tr><tr><td>SU8 developer</td><td>Micro resist technology GmbH, Germany</td><td>mr-Dev 600</td><td>-</td></tr><tr><td>Trimethylsilyl chloride&amp;nbsp;</td><td>Sigma-Aldrich, Switzerland</td><td>386529</td><td>&amp;ge;97%, CAUTION: Handle it only under fume hood.</td></tr><tr><td>Biopsy puncher</td><td>Miltex GmBH, Germany</td><td>33-31AA-P/25</td><td>1 mm</td></tr><tr><td>Biopsy puncher</td><td>Miltex GmBH, Germany</td><td>33-31A-P/25</td><td>1.5 mm</td></tr><tr><td>Glass coverslip</td><td>Menzel-Glaser, Germany</td><td>BB024040SC</td><td>24 mm &amp;times; 60 mm, #5</td></tr><tr><td>Laboratory Corona Treater</td><td>Electro-Technic Products, USA</td><td>BD-20ACV&amp;nbsp;</td><td>-</td></tr><tr><td>PTFE tubing</td><td>PKM SA, Switzerland</td><td>AWG-TFS-XXX</td><td>AWG 20TFS, roll of 100&amp;nbsp;m</td></tr><tr><td>Silicone rubber tubing</td><td>Hi-Tek Products, UK</td><td>-</td><td>1 mm I.D.</td></tr><tr><td>neMESYS Syringe Pumps</td><td>Cetoni GmbH, Germany</td><td>Low Pressure (290N)</td><td>-</td></tr><tr><td>High resolution camera</td><td>Zeiss, Germany</td><td>Axiocam MRc 5</td><td>-</td></tr><tr><td>Fluorescent inverted microscope</td><td>Zeiss, Germany</td><td>Axio Observer A1</td><td>Operable at two wavelengths, &lt;em&gt;i.e.&lt;/em&gt;, 350 nm and 488 nm</td></tr><tr><td>Green polystyrene fluorescent particles</td><td>Fisher Scientific, Switzerland</td><td>11523363</td><td>Size: 5.0 &amp;#956;m, solid content: 1%</td></tr><tr><td>Silver nitrate (AgNO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich, Switzerland</td><td>209139</td><td>&amp;ge;99.0%</td></tr><tr><td>L-Cysteine (Cys)</td><td>Sigma-Aldrich, Switzerland</td><td>W326305</td><td>&amp;ge;97.0%</td></tr><tr><td>Disposable weighing dish</td><td>Sigma-Aldrich, Switzerland</td><td>Z154881</td><td>L &amp;times; W &amp;times; H : 86&amp;nbsp;mm &amp;times; 86&amp;nbsp;mm &amp;times; 25&amp;nbsp;mm</td></tr><tr><td>Disposable weighing dish</td><td>Sigma-Aldrich, Switzerland</td><td>Z708593</td><td>Hexagonal, Size XL</td></tr><tr><td>Plastic spatula</td><td>Semadeni, Switzerland</td><td>3340</td><td>L &amp;times; W : 135 mm x 14 mm</td></tr><tr><td>Dye, Bemacron ROT E-G</td><td>Bezema, Switzerland</td><td>BZ 911.231</td><td>Red</td></tr><tr><td>Stereomicroscope</td><td>Wild Heerbrugg, Switzerland</td><td>Wild M8</td><td>500x magnification</td></tr><tr><td>Disposable scalpels</td><td>B. Braun, Switzerland</td><td>233-5320</td><td>Nr. 20</td></tr><tr><td>L-Ascorbic acid</td><td>Sigma-Aldrich, Switzerland</td><td>A4403</td><td>-</td></tr></tbody></table>

microfluidic pneumatic cages: a novel approach for in-chip crystal trapping, manipulation and controlled chemical treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

The double layer microfluidic devices consist of two bonded microfluidic chips structured in PDMS as shown in Figure 1. The first layer, which is at the same time bonded to a surface, is used to flow fluids (fluid layer), whilst the second layer, which is directly bonded to the first PDMS layer, is used to flow gas (control layer).
<img alt="Figure 1" src="/files/ftp_upload/54193/54193fig1.jpg" /> 
Figure 1. Double-layer microfluidic device. (A) Schematic illustration and (B) micrograph of the double-layer microfluidic device used in our investigations. <a href="https://www.jove.com/files/ftp_upload/54193/54193fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Injection of gas through channels in the control layer squeezes the fluid layer towards the surface (Figure 2A and Figure 2B), allowing trapping and localization of structures on the microfluidic channel surface. PDMS membrane actuation can be used to generate pneumatic cages and/or micro-valves that are controlled by a pneumatic controller. As exemplar models of membrane actuation, we show how the complete deflection of the fluid layer avoids a dye-laden flow to circulate underneath the valve after its actuation (Figure 2C) and trapping of fluorescent microparticles on the microchannel surface (Figure 2D and 2E).
<img alt="Figure 2" src="/files/ftp_upload/54193/54193fig2.jpg" /> 
Figure 2. Membrane actuation and trapping of structures. (A) Side and (B) top view illustrations showing the double-layer microfluidic device before (top) and after (bottom) actuation of the pneumatic valve. (C) Micrographs of a double-layer microfluidic device before (top) and after squeezing of the fluid layer (bottom). In bottom panel, the fluid layer is filled with an aqueous solution of rhodamine dye for a better perception of the membrane actuation. (D) Bright-field micrographs of a double-layer microfluidic device before (top) and after (bottom) actuation of the valve with a flowing aqueous solution containing polystyrene fluorescent particles (10 wt.%). (E) Fluorescent images of the optical microscope images shown in D. <a href="https://www.jove.com/files/ftp_upload/54193/54193fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3A illustrates the trapping of in situ generated CPs inside a double-layer microfluidic device through actuation of a pneumatic cage. Notice that a new coordination pathway is generated after the actuation of the first valve. The valve actuation ensures the trapping of the Ag(I)Cys CP generated at the interface of the two reagent streams and facilitates the formation of a new coordination pathway (Figure 3A). A detailed chemical characterization of the Ag(I)Cys CP generated at the interface of the two reagent streams can be found in previous studies17,18. Additionally, and after removal of the surplus reagents solutions with a flow of pure water (Figure 3B), a saturated ascorbic acid solution in ethanol can be added to the microfluidic channel for controlled chemical reduction of on-chip trapped structures (Figure 3C). Reducing the valve pressure from 3 bars to 1 bar favors a controlled chemical treatment of the trapped Ag(I)Cys CP underneath the clamped area18. The color change of trapped Ag(I)Cys CPs to dark brown is attributed to the reduction of monovalent silver to the metal, in accord with previous observations18,29.
<img alt="Figure 3" src="/files/ftp_upload/54193/54193fig3.jpg" /> 
Figure 3. Trapping of Ag(I)Cys CPs and controlled chemical reduction. (A) Optical microscope image showing the trapping of an in situ synthetized Ag(I)Cys CP and generation of a new coordination pathway. (B) Micrograph of trapped CPs underneath the clamped area after removal of surplus reagents solutions with a water flow, and in (C), micrograph of the same micro-valve after the reduction reaction process. <a href="https://www.jove.com/files/ftp_upload/54193/54193fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment at JoVE.com

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

microfluidic-pneumatic-cages-novel-approach-for-chip-crystal-trapping

Research

JoVE Journal

Chemistry

9.4K Views.  Empa - Swiss Federal Laboratories for Materials Science and Technology. Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures. 

Video: Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Bioengineering

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

Biology

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Engineering

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

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 Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. This platform has a three-dimensional (3D) network with curved fluid channels and three pneumatic valves, which create networks, channels, and spaces through duplex replication with polydimethylsiloxane (PDMS). The device operates based on the transient response of a fluid flow rate controlled by a pneumatic valve in the following order: (1) sample loading, (2) sample blocking, (3) sample concentration, and (4) sample release. The particles are blocked by thin diaphragm layer deformation of the sieve valve (Vs) plate and accumulate in the curved microfluidic channel. The working fluid is discharged by the actuation of two on/off valves. As a result of the operation, all particles of various magnifications were successfully intercepted and disengaged. When this technology is applied, the operating pressure, the time required for concentration, and the concentration rate may vary depending on the device dimensions and particle size magnification.

The present protocol describes a pneumatic microfluidic platform that can be used for efficient microparticle concentration.

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

Samenvatting

Meer video's verkennen