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...

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	<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. 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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>
			<td style="width:243px;"></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>
			<td style="width:243px;"></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>
			<td style="width:212px;"></td>
			<td style="width:112px;"></td>
			<td style="width:243px;"></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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		<tr height="21">
			<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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		<tr height="42">
			<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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		<tr height="23">
			<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 ...

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