Name: using adhesive patterning to construct 3d paper microfluidic devices
Uploaded: 2016-04-01T14:00:00
Description: Watch this Scientific Journal Video about Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices at JoVE.com

This work is supported by a fund from Bourns College of Engineering of University of California, Riverside. BK received a scholarship from the Lung-Wen Tsai Memorial Award in Mechanical Design.

1. Planar 4-layer Device (Stacked Layers) Construction
<ol>
	<li>Print arrays of each layer of the device9 onto each piece of filter paper using a solid ink printer.11,12 Place each filter paper on a hotplate at 170 &#176;C for 2 min. This will melt the wax-based ink and allow it to fully penetrate the thickness of the paper, forming hydrophobic barriers. 
	NOTE: The exact designs used are available as supplemental files.</li>
	<li>Remove filter paper from hotplate and allow it to cool to RT....

<ol>
	<li>Li, X., Ballerini, D. R., Shen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+paper-based+microfluidics:+Current+status+and+future+trends.">A perspective on paper-based microfluidics: Current status and future trends.</a> Biomicrofluidics. 6, 11301-11313 (2012).</li><li>Yetisen, A. K., Akram, M. S., Lowe, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+microfluidic+point-of-care+diagnostic+devices.">Paper-based microfluidic point-of-care diagnostic devices.</a> Lab Chip. 13, 2210-2251 (2013).</li><li>Cate, D. M., Adkins, J. A., Mettakoonpitak, J., Henry, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+paper-based+microfluidic+devices.">Recent developments in paper-based microfluidic devices.</a> Anal Chem. 87, 19-41 (2015).</li><li>Martinez, A. W., Phillips, S. T., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+microfluidic+devices+fabricated+in+layered+paper+and+tape.">Three-dimensional microfluidic devices fabricated in layered paper and tape.</a> Proc Natl Acad Sci U S A. 105, 19606-19611 (2008).</li><li>Fu, E., Ramsey, S. A., Kauffman, P., Lutz, B., Yager, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+in+two-dimensional+paper+networks.">Transport in two-dimensional paper networks.</a> Microfluid Nanofluidics. 10, 29-35 (2011).</li><li>Govindarajan, A. V., Ramachandran, S., Vigil, G. D., Yager, P., Bohringer, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low+cost+point-of-care+viscous+sample+preparation+device+for+molecular+diagnosis+in+the+developing+world;+an+example+of+microfluidic+origami.">A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami.</a> Lab Chip. 12, 174-181 (2012).</li><li>Schilling, K. M., Jauregui, D., Martinez, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper+and+toner+three-dimensional+fluidic+devices:+programming+fluid+flow+to+improve+point-of-care+diagnostics.">Paper and toner three-dimensional fluidic devices: programming fluid flow to improve point-of-care diagnostics.</a> Lab Chip. 13, 628-631 (2013).</li><li>Liu, H., Crooks, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+paper+microfluidic+devices+assembled+using+the+principles+of+origami.">Three-dimensional paper microfluidic devices assembled using the principles of origami.</a> J Am Chem Soc. 133, 17564-17566 (2011).</li><li>Lewis, G. G., DiTucci, M. J., Baker, M. S., Phillips, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+method+for+prototyping+three-dimensional,+paper-based+microfluidic+devices.">High throughput method for prototyping three-dimensional, paper-based microfluidic devices.</a> Lab Chip. 12, 2630-2633 (2012).</li><li>Kalish, B., Tsutsui, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+adhesive+enables+construction+of+nonplanar+three-dimensional+paper+microfluidic+circuits.">Patterned adhesive enables construction of nonplanar three-dimensional paper microfluidic circuits.</a> Lab Chip. 14, 4354-4361 (2014).</li><li>Carrilho, E., Martinez, A. W., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+wax+printing:+a+simple+micropatterning+process+for+paper-based+microfluidics.">Understanding wax printing: a simple micropatterning process for paper-based microfluidics.</a> Anal Chem. 81, 7091-7095 (2009).</li><li>Lu, Y., Shi, W., Jiang, L., Qin, J., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+of+paper-based+microfluidics+with+wax+for+low-cost,+portable+bioassay.">Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay.</a> Electrophoresis. 30, 1497-1500 (2009).</li><li>Maekawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genuine Japanese origami. , (2012).</li><li>Schonhorn, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+device+architecture+for+three-dimensional,+patterned+paper+immunoassays.">A device architecture for three-dimensional, patterned paper immunoassays.</a> Lab Chip. 14, 4653-4658 (2014).</li><li>Guan, J. J., He, H. Y., Hansford, D. J., Lee, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-folding+of+three-dimensional+hydrogel+microstructures.">Self-folding of three-dimensional hydrogel microstructures.</a> J Phys Chem B. 109, 23134-23137 (2005).</li></ol>

The above protocols use perforated metal sheets as stencils for applying aerosol adhesives to construct planar and nonplanar 3D paper microfluidic devices. In planar devices, this has the advantage of allowing devices to be completely unfolded after the adhesive has dried without destroying the device. In other adhesive based construction techniques, this is almost impossible, although, some designs allow for partial destructive disassembly by unpeeling two halves held together with a removable adhesive.14 Adh...

In recent years, paper microfluidics has garnered considerable popularity for its potential to provide low-cost point of care (POC) diagnostic devices.1-3 POC devices offer functionality similar to those of lab-based tests in a format that allows results to be obtained relatively quickly. POC devices made from paper are low-cost, lightweight, and easy-to-use alternatives to expensive microfluidic chips and miniaturized laboratories, making them ideal for use in resource-limited settings. The most common paper microfluidic devices are one-dimensional lateral flow devices, but planar three-dimensional (3D) paper microfluidic devices hold promise to provide multiplexed diagnostic devices4 that take up a much smaller footprint than would be required by a 2D device5 and correspondingly use a smaller sample volume.
Initially, planar 3D paper microfluidic devices were assembled individually, layer-by-layer with patterned paper layers alternating with laser-cut double-sided tape. Carefully aligned holes cut in the tape layer were filled with cellulose powder to ensure inter-layer fluid transport.4 A number of alternate methods were subsequently developed,6-9 each improving different aspects of the devices. In particular, by eschewing adhesives, devices could be folded via origami techniques with layers held together by an external clamp.8 This eliminates any potential adhesive interference in a diagnostic test and allows the device to be unfolded post-use, potentially allowing even smaller sample volumes by displaying results internally. Alternatively, by using an aerosol adhesive applied between each paper layer, sheets of devices could be assembled simultaneously, without time-consuming patterning and alignment of tape.9
However, by applying an aerosol adhesive through a stencil, it is possible to gain the benefit of both of these techniques. By spraying the adhesive through a stencil, only a fraction of the adhesive is applied to the device, minimizing any potential interference with interlayer fluid transfer. Additionally, with careful stencil selection, a pattern of adhesive can be applied that results in semi-permanent adhesive bonding, allowing devices to be unfolded after use, while still providing sufficient interlayer contact to allow fluid to wick between layers.
Finally, applying aerosol adhesives through a stencil eases the construction of nonplanar 3D paper microfluidic devices, by minimizing the amount of adhesive applied to adjacent faces that may require frequent folding and unfolding during construction.10 Additionally, the use of patterned adhesive enables device to be unfolded after use for more convenient storage. Nonplanar 3D paper microfluidic devices are expected to be used for tasks that would otherwise be impossible in a planar 3D device. Figure 1 depicts the general process flow used to construct both planar and nonplanar 3D devices.

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Camera</td>
			<td style="width:143px;">Nikon</td>
			<td style="width:119px;">D5100</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Solid-ink printer</td>
			<td style="width:143px;">Xerox</td>
			<td style="width:119px;">ColorQube 8880</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Hotplate</td>
			<td style="width:143px;">Torrey Pines</td>
			<td style="width:119px;">HS60</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Humidity chamber</td>
			<td style="width:143px;">Electro-Tech Systems</td>
			<td style="width:119px;">5503-E</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Spray adhesive</td>
			<td style="width:143px;">3M</td>
			<td style="width:119px;">62497749309</td>
			<td style="width:160px;">Super 77 (16.75 oz can)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Filter paper</td>
			<td style="width:143px;">Whatman</td>
			<td style="width:119px;">Grade 4</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Perforated steel sheet</td>
			<td style="width:143px;">MetalsDepot</td>
			<td style="width:119px;">PS16116</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Tartrazine</td>
			<td style="width:143px;">Sigma-Aldritch</td>
			<td style="width:119px;">T0388</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Allura Red</td>
			<td style="width:143px;">Sigma-Aldritch</td>
			<td style="width:119px;">458848</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:165px;">Erioglaucine disodium salt</td>
			<td style="width:143px;">Sigma-Aldritch</td>
			<td style="width:119px;">861146</td>
			<td style="width:160px;"></td>
		</tr>
	</tbody>
</table>

using adhesive patterning to construct 3d paper microfluidic devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

The 4-layer device tests were performed in a sealed chamber, shielding them from any wind or breezes that might cause excessive evaporation of the limited deposited fluid volume. The majority of the wicking in the 4-layer devices is in the middle layers of the device, so differences in wicking speeds due to evaporation were expected to be minimal. Additionally, there is minimal lateral wicking, with only 13 mm between the inlet and any individual outlet, suggesting that variations in wick...

Watch this Scientific Journal Video about Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices at JoVE.com

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

The overall goal of this technique is to create planar and nonplanar three-dimensional paper microfluidic devices that are able to be unfolded during construction and after use through the patterning of aerosol adhesive. This method opens up a brand new design space for paper microfluidics, by removing constraints which previously kept researchers from fabricating out of plain channel networks. The primary advantages of this technique are that it significantly reduces the amount of adhesive applied during the fabrication of paper microfluidic devices, and that it enables the construction of nonplanar three-dimensional paper microfluidic circuits.First, print an array of each layer for the device onto filter paper using a solid ink printer. Place each filter paper on a hot plate at 170 degrees Celsius for two minutes, to melt the wax-based ink, and allow it to fully penetrate into the paper, forming hydrophobic barriers. Once the paper has cooled, put a different dye color in each of the branches of layer three using a micropipette.Four microliter aliquots of five millimolar dyes will suffice. Now, begin the construction. First, clamp the bottommost layer between the stencil and a stiff backing, such as a piece of plate glass.Ensure that the stencil is flat against the paper to minimize spray shadows. Then, set a metronome to 180 beats per minute and spray adhesive from approximately 24 centimeters away for four beats. Move the can across the stencil in four even motions.If the can is moved too slowly, the adhesive will accumulate on the stencil itself, clogging the stencil. If the can is moved too rapidly, not enough adhesive will be applied. Next, remove the stencil and position the next layer of the device over the freshly-sprayed layer, carefully aligning them at the edges.Firmly press the layers together. Then, spray the adhesive again. Continue this process until all the layers are tightly adhered.Onto the four-layer stack, stick a strip of packing tape across the bottom layer to prevent fluid from leaking out. Then, individual devices can be cut from the stack following the printed pattern. Like the previous procedure, print the device onto filter paper using a solid ink printer and melt the ink on a hot plate at 170 degrees Celsius for two minutes.For this procedure, also print the crease pattern in the same manner, but on regular printer paper. Once the prints have cooled, align the lines of the crease pattern to the edges of the channel patterns. Then, secure them using tape.Next, trace the crease pattern with a blunt stylus. Apply enough force to mark the device sheet but do not tear the paper. If a tear occurs, start over.This pre-creasing technique increases the accuracy and precision of the folding. Now, begin folding the device using mountain and valley folds according to the crease pattern. Folding before application of the adhesive helps speed the device assembly.Once folded, unfold it to expose the portions that require adhesive. Then, with a blade, cut out masks to limit where adhesive will get applied. Now, clamp the device flatly between the stencil with the mask and a stiff backing.Use a metronome to time out 1.3 seconds and apply the adhesive as before. If the ambient humidity is low, apply the adhesive in a humidity-controlled area so the adhesive doesn't dry too quickly. Next, remove the stencil and mask and turn the sheet over.Then, spray the back side of the paper in the same manner. Immediately remove the device from the stencil and begin folding the device. Once the device is completely folded, apply continuous pressure to the adhesive-containing portion until it is dried.To perform a wicking test on the four-layer devices, randomly select 20 devices. Place the devices where they are shielded from air currents to minimize evaporation. Then, deposit 40 microliters of water at the inlet of each device.Record the time it takes for each device to have all of its outlets completely filled with dye. For the origami devices, compare two origami peacocks, one made as previously described, and the other made without using the stencil while applying the adhesive. Then, insert one end of a small paper lead into the body of the peacock.Under controlled relative humidity, above 90%place each leg and the paper lead of each peacock into a container filled with five millimolar dye. Average wicking times and success rates for four-layer devices constructed with different amounts of applied adhesive were compared. Uniform adhesive coverage resulted in relatively high success rates that decreased with increasing quantities of adhesive.Success rates were also much higher with faster wicking times when the patterned adhesive was applied to both sides, than only one side. Failures occurred more frequently when adhesive was only applied to one side. Typical stacked device failure was characterized by outlets that failed to completely fill with dye or took longer than five minutes to fill.In origami folded devices, device failure was characterized by outlets that failed to fill with any amount of dye. These outlets were exclusively located along the two edges of the device that contained the creases. By doubling the size of the border around the channels, the success rates for both single and dual-sided adhesive applications increased.Both methods of adhesive application resulted in devices that successfully routed liquid the length of their channels and without mixing. However, the device with uniformly-applied adhesive was noticeably slower. While attempting this procedure, it's important to ensure even and consistent adhesive application.Do note that aerosol adhesive should only be applied in well-ventilated areas. After watching this video, you should have a good understanding of how to apply and use patterned adhesives to construct planar and nonplanar 3D paper microfluidic devices. This technique will allow researchers to explore the use of nonplanar structures to achieve functionality not previously found in planar devices, such as integrated actuation and sensing.

using-adhesive-patterning-to-construct-3d-paper-microfluidic-devices

Research

JoVE Journal

Bioengineering

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...