Name: soft lithographic procedure for producing plastic microfluidic devices with view-ports transparent to visible and infrared light
Uploaded: 2017-08-17T13:00:00
Description: Watch this Scientific Journal Video about Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light at JoVE.com

The authors gratefully acknowledge MBI financial support.

1. Preparation of Silicon Primary Mold
NOTE: A photomask is required for the preparation of the primary mold. The photomask can be either purchased from independent providers or fabricated in-house through standard optical mask fabrication procedures. A photomask with bright field polarity is used in this case (Figure 2a).
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	<li>Pattern definition
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		<li>Spin coat a 4 inch silicon wafer with SU-8 3010 negative photoresi...

<ol>
	<li>Holman, H. -. Y. N., 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=Synchrotron+infrared+spectromicroscopy+as+a+novel+bioanalytical+microprobe+for+individual+living+cells:+cytotoxicity+considerations.">Synchrotron infrared spectromicroscopy as a novel bioanalytical microprobe for individual living cells: cytotoxicity considerations.</a> BIOMEDO. 7 (3), 417-424 (2002).</li><li>Liu, K. Z., Xu, M., Scott, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+characterisation+of+leucocytes+by+infrared+spectroscopy.">Biomolecular characterisation of leucocytes by infrared spectroscopy.</a> Br J Haematol. 136 (5), 713-722 (2007).</li><li>Moss, D. A., Keese, M., Pepperkok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IR+microspectroscopy+of+live+cells.">IR microspectroscopy of live cells.</a> Vibrational Spectroscopy. 38 (1-2), 185-191 (2005).</li><li>Rahmelow, K., Hubner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+Spectroscopy+in+Aqueous+Solution:+Difficulties+and+Accuracy+of+Water+Subtraction.">Infrared Spectroscopy in Aqueous Solution: Difficulties and Accuracy of Water Subtraction.</a> Appl Spectrosc. 51 (2), 160-170 (1997).</li><li>Kazarian, S. G., Chan, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR-FTIR+spectroscopic+imaging:+recent+advances+and+applications+to+biological+systems.">ATR-FTIR spectroscopic imaging: recent advances and applications to biological systems.</a> Analyst. 138 (7), 1940-1951 (2013).</li><li>Loutherback, K., Chen, L., Holman, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-channel+microfluidic+membrane+device+for+long-term+FT-IR+spectromicroscopy+of+live+adherent+cells.">Open-channel microfluidic membrane device for long-term FT-IR spectromicroscopy of live adherent cells.</a> Anal Chem. 87 (9), 4601-4606 (2015).</li><li>Loutherback, K., Birarda, G., Chen, L., Holman, H. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+approaches+to+synchrotron+radiation-based+Fourier+transform+infrared+(SR-FTIR)+spectral+microscopy+of+living+biosystems.">Microfluidic approaches to synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy of living biosystems.</a> Protein Pept Lett. 23 (3), 273-282 (2016).</li><li>Dousseau, F., Therrien, M., P&#233;zolet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Spectral+Subtraction+of+Water+from+the+FT-IR+Spectra+of+Aqueous+Solutions+of+Proteins.">On the Spectral Subtraction of Water from the FT-IR Spectra of Aqueous Solutions of Proteins.</a> Appl Spectrosc. 43 (3), 538-542 (1989).</li><li>Tobin, M. J., 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=FTIR+spectroscopy+of+single+live+cells+in+aqueous+media+by+synchrotron+IR+microscopy+using+microfabricated+sample+holders.">FTIR spectroscopy of single live cells in aqueous media by synchrotron IR microscopy using microfabricated sample holders.</a> Vibrational Spectroscopy. 53 (1), 34-38 (2010).</li><li>Birarda, G., 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=Infrared+microspectroscopy+of+biochemical+response+of+living+cells+in+microfabricated+devices.">Infrared microspectroscopy of biochemical response of living cells in microfabricated devices.</a> Vibrational Spectroscopy. 53 (1), 6-11 (2010).</li><li>Vaccari, L., Birarda, G., Businaro, L., Pacor, S., Grenci, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+microspectroscopy+of+live+cells+in+microfluidic+devices+(MD-IRMS):+toward+a+powerful+label-free+cell-based+assay.">Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.</a> Anal Chem. 84 (11), 4768-4775 (2012).</li><li>Mitri, 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=SU-8+bonding+protocol+for+the+fabrication+of+microfluidic+devices+dedicated+to+FTIR+microspectroscopy+of+live+cells.">SU-8 bonding protocol for the fabrication of microfluidic devices dedicated to FTIR microspectroscopy of live cells.</a> Lab Chip. 14 (1), 210-218 (2014).</li><li>Birarda, G., 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=IR-Live:+fabrication+of+a+low-cost+plastic+microfluidic+device+for+infrared+spectromicroscopy+of+living+cells.">IR-Live: fabrication of a low-cost plastic microfluidic device for infrared spectromicroscopy of living cells.</a> Lab Chip. 16 (9), 1644-1651 (2016).</li><li>Wehbe, K., Filik, J., Frogley, M. D., Cinque, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+optical+substrates+on+micro-FTIR+analysis+of+single+mammalian+cells.">The effect of optical substrates on micro-FTIR analysis of single mammalian cells.</a> Anal Bioanal Chem. 405 (4), 1311-1324 (2013).</li><li>Helmut, S., 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=Controlled+co-evaporation+of+silanes+for+nanoimprint+stamps.">Controlled co-evaporation of silanes for nanoimprint stamps.</a> Nanotechnology. 16 (5), 171 (2005).</li><li>Loewen, E. G., Popov, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diffraction Gratings and Applications. , (1997).</li><li>. Technical Note: Optical Materials Available from: <a target="_blank" href="https://www.newport.com/n/optical-materials">https://www.newport.com/n/optical-materials</a> (2017)</li><li>Cai, D., Neyer, A., Kuckuk, R., Heise, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman,+mid-infrared,+near-infrared+and+ultraviolet-visible+spectroscopy+of+PDMS+silicone+rubber+for+characterization+of+polymer+optical+waveguide+materials.">Raman, mid-infrared, near-infrared and ultraviolet-visible spectroscopy of PDMS silicone rubber for characterization of polymer optical waveguide materials.</a> J Mol Struc. 976 (1-3), 274-281 (2010).</li></ol>

In order to assess and optimize the fabrication protocol, we used a simple layout for the microfluidic pattern with a large rectangular chamber (5 mm x 2.5 mm size) at the center, two small rectangular chambers (5.5 mm x 0.75 mm size) separated from the main circuit on the upper and lower sides, and 300 &#181;m wide in-let/out-let channels. The central chamber is used for the seeding and observation of the cells, while the two separated smaller chambers are used to measure the air background during FTIR experiments as a ...

Fourier Transform Infrared Spectro-microscopy (FTIR) has been extensively utilized as a label-free and non-invasive imaging technique to provide detailed chemical information of a sample. This enables the extraction of biochemical information to study the chemistry of biological samples, with a minimum amount of preparation since the absorption spectrum of the specimen carries the intrinsic fingerprints of its chemical composition1,2. Recently, FTIR has been increasingly applied to the study of live biological samples, e.g., cells3. However, water, which is the medium for living cells in most cases, shows a strong absorbance in the mid-IR region. Even as a thin layer, its presence can completely overwhelm the important structural information of the specimens.
For many years, the common approach was fixing or drying samples to completely exclude the water absorption signal in the spectrum. However, this approach does not allow for real time measurements on living cells, which is essential to study the change of their chemical composition and cellular processes with time. One way to obtain reliable absorption spectra from live biological samples, is to limit the total optical path length in the medium of the IR beam to less than 10 &#181;m4.
A well-established approach in living cell experiments has been so far, Attenuated Total Reflection (ATR)-FTIR imaging, which enables measurements independent of the sample thickness, allowing cells to be sustained in a thicker layer of aqueous medium. However, the small depth of penetration of the evanescent wave restricts measurements of samples to only the first few microns from the surface of the ATR crystal5.
Alternatively, the water absorption limitation has been circumvented with the emergence of various microfluidic systems, which are generally classified into two large groups: open channel (where one of the fluid surfaces is exposed to the atmosphere) and closed channel (where two IR-transparent windows are separated by a spacer with a defined thickness).
Loutherback et al. developed an open-channel membrane device that enables long term continuous IR measurements of live cells for up to 7 days6. The method requires high humidity in the environment to prevent evaporation of the medium from the cell surface. The system works best with cells that naturally grow at air-liquid interfaces, such as epithelial tissues of the skin, lung, and eyes, or microbial biofilms7.
A closed-channel configuration aims to create a uniform, thin layer between two parallel IR-transparent windows, where cells are maintained in their aqueous media. The thickness of this cavity is such that the water absorption signal is below saturation. Water background can then be subtracted to obtain the correct sample spectra. Most of the closed-channel methods utilize a plastic spacer separating the two windows to form a demountable liquid chamber3,8,9. An advantage of this method is that it does not require microfabrication; however, structures that are more complex than a measuring chamber with in- and out-let channels are extremely difficult to realize in the thin spacer. There is also an issue with the reproducibility of the path length between IR measurements due to its reliance on mechanical clamping. In order to achieve a more precise control of the spacing for a more reliable spectrum acquisition, optical lithography methods have been implemented to pattern photoresist on top of the IR substrate to define the spacer9,10,11,12. Even though this makes it possible for more complex structures to be defined in the spacer, the method requires access to a microfabrication facility to produce the pattern on every substrate.
In this paper, we present a simple fabrication technique of an IR-compatible microfluidic device, with the aim to reduce the fabrication cost and the requirement of accessing a microfabrication facility. The method presented here (see Figure 1) uses an established process known as soft lithography. Two molds are required in this case. The primary mold is made from a 4-inch silicon wafer using a standard UV lithography process. The secondary mold is its replica made of PDMS, which has a reversed polarity of the pattern in the silicon primary mold and serves as the master mold for subsequent device fabrication.
The device has two separate layers: a first layer with the microfluidic layout (which in the presented case consists of the microfluidic channel, in-let/out-let, and an observation chamber with a CaF2 viewport), and a second layer with a flat surface (which consists of only a CaF2 viewport).
Here an UV-curable optical adhesive, Norland optical adhesive 73 (NOA73, abbreviated as NOA henceforth), is used to form the main plastic body of the device. There are several advantages of using this optical adhesive: low fabrication cost, ease of connectivity to external systems, good optical transparency, low viscosity, and most importantly, biocompatibility13. CaF2 is a suitable choice as the viewport due to its biocompatibility and excellent IR-transparency14.
With this new approach, access to a microfabrication facility is strictly required only for the fabrication of the primary mold. Subsequent fabrication processes for the plastic microfluidic device can be carried out in any laboratory equipped with an UV-illumination source.

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			<td height="21" style="height:21px;width:291px;">Chemical</td>
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			<td height="42" style="height:42px;width:291px;">Trichloro(1H,1H,2H,2H-perfluorooctyl)silane 97%</td>
			<td style="width:212px;">Sigma Aldrich</td>
			<td style="width:112px;">448931-10G</td>
			<td style="width:243px;"></td>
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			<td height="42" style="height:42px;width:291px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:212px;">Dow Corning</td>
			<td style="width:112px;"></td>
			<td style="width:243px;">Polydimethylsiloxane or in short, PDMS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Norland Optical Adhesive 73</td>
			<td style="width:212px;">Norland Products Inc.</td>
			<td style="width:112px;">7304</td>
			<td style="width:243px;"></td>
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			<td height="21" style="height:21px;width:291px;">SU8 3010 photoresist</td>
			<td style="width:212px;">MicroChem</td>
			<td style="width:112px;">Y311060</td>
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			<td height="21" style="height:21px;width:291px;">SU8 developer</td>
			<td style="width:212px;">MicroChem</td>
			<td style="width:112px;">Y020100</td>
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			<td height="21" style="height:21px;width:291px;">Material</td>
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			<td height="21" style="height:21px;width:291px;">Silicon wafer, 4 inch, prime grade</td>
			<td style="width:212px;">Bonda Technology Pte Ltd</td>
			<td style="width:112px;"></td>
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			<td height="21" style="height:21px;width:291px;">CaF2 IR-grade windows</td>
			<td style="width:212px;">Crystran, UK</td>
			<td style="width:112px;">CAFP10-1</td>
			<td style="width:243px;">10 mm diameter, 1 mm thickness</td>
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			<td height="21" style="height:21px;width:291px;">Acrylic templates</td>
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			<td style="width:243px;">Custom made</td>
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			<td height="21" style="height:21px;width:291px;">Equipment</td>
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			<td height="21" style="height:21px;width:291px;">UV-KUB 2 (UV LED exposure system)</td>
			<td style="width:212px;">KLOE</td>
			<td style="width:112px;"></td>
			<td style="width:243px;">Emission spectrum 365nm &#177; 5nm</td>
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			<td height="21" style="height:21px;width:291px;">Newport UV lamp</td>
			<td style="width:212px;">Newport</td>
			<td style="width:112px;">Model 66902</td>
			<td style="width:243px;">50-500 Watt Hg arc lamp</td>
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			<td height="21" style="height:21px;width:291px;">CEE Spin coater</td>
			<td style="width:212px;">Brewer Science</td>
			<td style="width:112px;">Model 200x</td>
			<td style="width:243px;"></td>
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			<td height="21" style="height:21px;width:291px;">MJB4 mask aligner</td>
			<td style="width:212px;">SUSS MicroTec</td>
			<td style="width:112px;"></td>
			<td style="width:243px;"></td>
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			<td height="21" style="height:21px;width:291px;">Precision digital hot plate</td>
			<td style="width:212px;">Harry Gestigkeit GmbH</td>
			<td style="width:112px;">2860SR</td>
			<td style="width:243px;"></td>
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			<td height="42" style="height:42px;width:291px;">Plasma Surface Technology</td>
			<td style="width:212px;">Diener Electronic GmbH + Co. KG</td>
			<td style="width:112px;"></td>
			<td style="width:243px;">For O2 plasma treatment</td>
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			<td height="23" style="height:23px;width:291px;">IDP-3 Dry Scroll Vacuum Pump</td>
			<td style="width:212px;">Agilent Technologies</td>
			<td></td>
			<td>ultimate pressure 3.3 x 10-1 mbar</td>
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			<td height="21" style="height:21px;width:291px;">Bruker IFS 66v/s FTIR Spectrometer</td>
			<td style="width:212px;">Bruker</td>
			<td></td>
			<td></td>
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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.

Figure 6 presents the transmittance spectra of a brand new CaF2 window, the patterned half of the device, and the complete device. All three spectra exhibit excellent transparency to mid IR with transmittance larger than 80%. The interference pattern visible in the spectrum of the full device (yellow curve in the figure) is caused by the air gap in the range of 9-10 &#181;m between the two windows. These spectra demonstrate that the fabrication app...

Watch this Scientific Journal Video about Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light at JoVE.com

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

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 ...

The over goal of this microfabrication protocol is to produce plastic microfluidic devices compatible with Fourier transform infrared microspectroscopy in a simple and cost effective way. These methods can help with vast understanding of cellular biochemistry. It provides us simplified access to infrared microspectroscopy, a label free, no damaging imaging technique which can retrieve biochemical maps of live cells.The main advantage of the this technique is that it reduces the need for accessing micro-fabrication facility and it uses plastic as the main component for the final devices. Demonstrating the procedure will be Mona Suryana, a research assistant from our laboratory. To begin this procedure, prepare the silicon primary mold as outlined in the text protocol.Next, mix the PDMS elastomer and the curing agent at 10 to one ratio. The total amount mixed is such that the resulting PDMS is approximately one to 1.5 millimeters in thickness. After a thorough mixing, transfer the mixture to a vacuum jar.Lower the pressure until it is between one and 10 millibar. Wait for 15 minutes or until there are no visible bubbles to degas the mixture. Pour the degassed PDMS onto the prepared silicon mold.Transfer the mold to the vacuum jar and lower the pressure for 15 minutes to degas the mixture once again. Then transfer the mold to a hot plate or oven. Heat the mold at 70 degrees Celsius for two hours to cure the mixture.Remove the cured PDMS from the heat and let it cool to room temperature. Use a razor blade to cut the PDMS along the edges of the silicon mold. Using a pair of tweezers, pinch one corner, and carefully peel the PDMS replica off the silicon mold.Next, transfer the PDMS mold to a plasma cleaner. Set the chamber pressure between one and 10 millibar and treat the PDMS mold with oxygen plasma at 60 watts for 30 seconds with 20 SCCM of oxygen flow. After this, place the mold in a vacuum jar and add about 50 microliters of silane.Leave the jar in the vacuum state for two hours. To begin, design or acquire acrylic templates, as outlined in the text protocol and prepare PDMS elastomer and curing agent as shown at the beginning of this procedure. Pour the degassed PDMS mixture on the acrylic templates until the topmost surface is submerged, about one millimeter below the liquid surface.Then transfer the submerged templates to a vacuum jar. Degas the PDMS using the same process as before. Then transfer the submerged templates to a hot plate or an oven.Heat at 60 degrees Celsius for two hours to cure the mixture. Remove the cured PDMS from the heat source and let it cool to room temperature. Using a razor blade, cut the PDMS along the edges of the acrylic templates.Next use a pair of tweezers to pinch one corner and carefully peel the PDMS off. After this, prepare the second PDMS replica as outlined in the text protocol. To begin fabricating the patterned half of the device, treat a calcium fluoride window with oxygen plasma, at 60 watts for 30 seconds, with 20 SCCM of oxygen flow.Carefully place the first PDMS template, the one with the small pillars, onto a flat surface. Then, place the treated calcium fluoride window in the center of the template. Press gently to ensure the window is fully in contact with the PDMS.Next, place a UV transparent plate on the backside of the PDMS mold, aligned with the location of the central chamber. Press gently to ensure it is fully in contact with the PDMS. Place the mold onto the PDMS template with the fluidic pattern face down and with the fluidic chamber aligned to the center of the window.Then gradually dispense drops of NOA at the inlet of the PDMS template. Allow the NOA to slowly fill the cavity. After the cavity is completely filled, cure the NOA by exposing the mold to UV light.Carefully remove the UV transparent plate from the mold. Gently peel the PDMS mold from the top of the NOA layer. After this remove the NOA layer.To begin fabricating the flat half of the device, treat the calcium fluoride window with oxygen plasma at 60 watts for 30 seconds with 20 SCCM of oxygen flow. Carefully place the second PDMS template, the one without the small pillars, onto a flat surface. Place the treated calcium fluoride window in the center of template.Press gently to ensure the window is fully in contact with the PDMS. Next acquire a one millimeter thick sheet of PDMS that is five centimeters by 3.5 centimeters. Place this sheet on top of the calcium fluoride window, aligned with the center of the template.Press gently to ensure the sheet is fully in contact with the window. Gradually dispense drops of NOA at the inlet of the PDMS template. Allow the NOA to slowly fill the cavity.After the cavity is completely filled, cure the NOA by exposing the mold to UV light. Peel off the PDMS layer. Then carefully remove the cured NOA layer from the PDMS template.Place one half of the device on top of the other, with the calcium fluoride windows aligned. Gently press at the corners of the NOA layers, fixing the position of the two halves. Next, acquire PDMS discs and rectangles as outlined in the text protocol.Place the PDMS discs in the corresponding openings of the device. Next, place the PDMS rectangles with precut openings onto each side. Transfer the entire assembly to the vacuum press sandwiching it between two plates.Then, seal the plastic bag. Turn on the vacuum pump to evacuate the assembly and allow it to run for at least 10 minutes. Using a broadband mercury vapor lamp at 270 watts, expose the evacuated assembly to UV light for 15 minutes.After this, turn off the vacuum pump and let the assembly slowly vent to atmospheric pressure before removing the final device from the assembly. In this procedure, a plastic microfluidic device with view-ports transparent to visible and infrared light is fabricated. Transmittance spectra are then acquired to compare a brand new calcium fluoride window, one half of the fabricated device, and the complete device.As can be seen, all three exhibit a transmittance over 80%up to mid-infrared, indicating a high degree of transparency in this range. While the spectrum for the full device exhibits an interference pattern caused by the air gap between the two windows, these spectra demonstrate that the fabrication process does not alter the transparency of calcium fluoride windows up through the mid-infrared range. Once mastered, this technique can be done in one hour if it is performed properly and the required templates and molds are ready.After watching this video, you should have good understanding of how to produce plastic microfluidic devices compatible with Fourier transform infrared spectroscopy using replica molding with PDMS and the capillarity filling process with UV curable resins. Don't forget that working with with silane and UV light can be extremely hazardous and always conduct procedure-based risk assessments and wear appropriate person protective equipment while performing this procedure.

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Research

JoVE Journal

Bioengineering

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

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 ...

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 ...

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.

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 ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

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 ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.