Name: Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light (Video) | JoVE Journal
Uploaded: 2017-08-17T13:00:00.000+00:00
Duration: 10 min 26 s
Description: Learn soft lithography for fabricating plastic microfluidic devices compatible with Fourier transform infrared microspectroscopy. This cost-effective method enables label-free biochemical imaging of live cells without damaging samples.

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5 minutes

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).
<ol>
	<li>Pattern definition
	<ol>
		<li>Spin coat a 4 inch silicon wafer with SU-8 3010 negative photoresist at 2,300 rpm for 30 s.</li>
		<li>Soft-bake the photoresist on a hot plate at 65 &#176;C for 2 min and then at 95 &#176;C for 8 min.</li>
		<li>Expose the photoresist to UV light (i-line, 365 nm) through the photomask under the mask aligner for a total energy dose of 100-120 mJ/cm2; the hard contact mode is preferred to achieve a better resolution.</li>
		<li>Remove the wafer and apply a post exposure bake at 65 &#176;C for 1 min and then at 95 &#176;C for 2 min.</li>
		<li>Develop the photoresist using a SU-8 developer at room temperature, then rinse with isopropyl alcohol, and gently blow dry with nitrogen; the measured pattern thickness should be 10 &#181;m and below. See Figure 2b for the picture of the actual silicon mold.</li>
	</ol>
	</li>
	<li>Silanization of silicon mold
	<ol>
		<li>Treat the silicon mold with oxygen plasma at 60 W for 30 s with 20 sccm of oxygen flow. Set the chamber pressure to 1-10 mbar during the process.</li>
		<li>Place the mold in a vacuum jar with 50 &#181;L of silane and leave the jar in the vacuum state (1-10 mbar) for at least 2 h. 
		NOTE: The silanization process creates a hydrophobic surface coating which prevents PDMS from sticking to the mold15. Note that the primary mold can also be fabricated using an alternative method, which involves dry etching of the silicon. In this case, the photomask will be of opposite polarity (dark field), and the pattern definition in step 1.1 will use a positive photoresist.</li>
	</ol>
	</li>
</ol>
2. Preparation of PDMS Secondary Mold
<ol>
	<li>PDMS mixing
	<ol>
		<li>Mix the PDMS elastomer and curing agent, 10:1; the total amount is such that the resulting PDMS thickness is approximately 1 to 1.5 mm.</li>
		<li>After thorough mixing, degas the mixture by leaving it in a vacuum jar in the vacuum state (1-10 mbar) for approximately 15 min or until there are no visible bubbles; this is to remove any trapped air within the mixture.</li>
	</ol>
	</li>
	<li>Mold replication
	<ol>
		<li>Pour the PDMS mixture on the silicon mold fabricated in step 1 and degas the mixture to remove any trapped air with the same settings as in step 2.1.2. Heat at 70 &#176;C for 2 h on a hot plate to cure the mixture.</li>
		<li>Remove the cured PDMS from the hot plate and let it cool down to room temperature. With a razor blade, cut the PDMS along the edges of the silicon mold.</li>
		<li>With a pair of tweezers, pinch one corner of the cut PDMS and carefully peel the PDMS replica off the silicon mold; the resulting microfluidic pattern on this secondary mold is a protrusion, which is the opposite polarity of the primary mold (Figure 2c).</li>
	</ol>
	</li>
	<li>Silanization of the PDMS replica (same as in step 1.2)
	<ol>
		<li>Treat the PDMS mold with oxygen plasma at 60 W for 30 s with 20 sccm of oxygen flow. Set the chamber pressure to 1-10 mbar during the process.</li>
		<li>Place the mold in a vacuum jar with 50 &#181;L of silane and leave the jar in the vacuum state (1-10 mbar) for at least 2 h.</li>
	</ol>
	</li>
</ol>
3. Preparation of PDMS Templates
NOTE: To standardize the shape and size of the final devices and to ease the alignment of the main features in the two halves, two separate PDMS templates were used, which define the geometry of the device, the placement of the transparent window and the in- and out-let connections. The first PDMS template aids the fabrication of the patterned half of the device, while the second helps to ease the fabrication of the flat half of the device.
<ol>
	<li>Design the templates using a computer aided design (CAD) software. Figure 3a shows the lay-out and dimensions of the template used to fabricate the patterned half of the device. To fabricate the flat half of the device, remove the 1.5 mm diameter holes from the design.</li>
	<li>Acquire the templates from an external provider or through an internal mechanical workshop if available. 
	NOTE: Acrylic was used as the template material due to ease of fabrication and low cost achievable in any standard workshop. There are alternative options, such as 3D printing.</li>
	<li>Mix the PDMS elastomer and curing agent 10:1; be sure to prepare a sufficient amount of PDMS to entirely submerge the templates.</li>
	<li>After thorough mixing, degas the mixture by leaving it in a vacuum jar in the vacuum state (1-10 mbar) for approximately 15 min or until there are no visible bubbles (whichever is later); this is to remove any trapped air within the mixture.</li>
	<li>Pour the PDMS mixture on the acrylic templates until their top-most surface is submerged about 1 mm below the liquid surface. Degas the PDMS again to remove any trapped air with the same settings as in 3.4. Heat this at 60 &#176;C for 2 h on a leveled hot plate to cure the mixture.</li>
	<li>Remove the cured PDMS from the hot plate and let it cool down to room temperature. With a razor blade, cut the PDMS along the edges of the acrylic templates.</li>
	<li>With a pair of tweezers, pinch one corner of the cut PDMS and carefully peel the PDMS off the acrylic templates. 
	NOTE: Figure 3b shows the lay-out and dimensions of the PDMS replica used to fabricate the patterned half of the device.</li>
	<li>Prepare the second PDMS replica for fabricating the flat half of the device by repeating steps 3.3 to 3.7 but using the acrylic template without the 1.5 mm diameter holes.</li>
</ol>
4. Microfluidic Device Fabrication
<ol>
	<li>Fabrication of the patterned half of the device (i.e. , with device layout)
	<ol>
		<li>Treat the CaF2 window with oxygen plasma at 60 W for 30 s with 20 sccm of oxygen flow. This is done to improve the flow of NOA during the following fabrication. 
		NOTE: This step is not mandatory.</li>
		<li>Place carefully the first PDMS template (one with the 1.5 mm diameter pillars) on a flat surface, e.g., a soda lime plate (Figure 4a). Place a CaF2 window centered on top of the PDMS plug and gently press the window such that it is in good contact with the plug (Figure 4b).</li>
		<li>Take the PDMS mold made in step 2 and place a thin UV-transparent plate (in this case, a quartz plate, 500 &#181;m thick and 1.5 cm x 1.5 cm in size) on the backside of the mold, aligned with the location of the central chamber (Figure 4c). Make sure that the quartz plate is in good contact with the PDMS mold. 
		NOTE: The quartz plate prevents the unwanted area of the mold from easily coming into contact with the CaF2 window.</li>
		<li>Gently place this PDMS mold face down towards the CaF2 window with the fluidic chamber aligned to the center of the CaF2 window. Make sure that all the elements (template, mold and window) are in good contact and aligned (Figure 4c-4d).</li>
		<li>Gradually dispense drops of NOA at the in-let of the PDMS template and let it slowly fill the cavity. Once the resin comes into contact with the edge of the window, the capillary flow will fill the thin gap (~10 &#181;m) between the PDMS mold and CaF2 window (Figure 4e-4f).</li>
		<li>After the cavity is completely filled, cure the NOA by exposure to UV light (e.g., with a UV-LED exposure system, Figure 4g). 
		NOTE: The exposure time may vary accordingly with the energy of the UV source. The UV-LED exposure system, which provides a power density of 24 mW/cm2, requires around 90 s at 100% power and continuous exposure mode.</li>
		<li>Carefully remove the thin quartz plate from the back of the PDMS mold and then gently peel the PDMS mold from the top of the NOA layer (Figure 4h). Finally, remove the NOA layer from the PDMS template (Figure 4i). 
		NOTE: The resulting device layout on the cured NOA would have the same polarity of the pattern in the primary silicon mold.</li>
	</ol>
	</li>
	<li>Fabrication of the flat half of the device (i.e. , without device layout)
	<ol>
		<li>Treat the CaF2 window with oxygen plasma at 60 W for 30 s with 20 sccm of oxygen flow. 
		NOTE: This step is not mandatory.</li>
		<li>Place carefully the second PDMS template (one without the 1.5 mm diameter pillars) on a flat surface, e.g., a soda lime plate. Place a CaF2 window centered on top of the PDMS plug and gently press the window such that it is in good contact with the plug.</li>
		<li>Place a 1 mm thick PDMS sheet with 5 cm x 3.5 cm size on top of the CaF2 window, with the PDMS sheet aligned with the center of the PDMS template. Make sure that the PDMS sheet is in good contact with the window.</li>
		<li>Gradually dispense drops of NOA at the in-let of the PDMS template and let it slowly fill the cavity.</li>
		<li>After the cavity is completely filled, cure the NOA by exposure to UV light (e.g., with a UV-LED exposure system). 
		NOTE: The exposure time may vary accordingly with the UV energy source. With the UV-LED exposure system, which provides a power density of 24 mW/cm2, this requires around 50 s at 100% power and continuous exposure mode.</li>
		<li>Peel off the PDMS sheet from the top of the NOA layer and carefully remove the cured NOA layer from the PDMS template.</li>
	</ol>
	</li>
	<li>Bonding of the two halves of the device
	<ol>
		<li>Align the two halves of the device such that both CaF2 windows are aligned. Gently finger-press both halves at the corner of the NOA layers such that the position of the two halves are fixed.</li>
		<li>Cut two circular discs out of a 1 mm thick PDMS sheet using an 8 mm diameter puncher (Figure 5a).</li>
		<li>Cut two rectangles with the same size of the device (4 cm x 2.5 cm) from a 1 mm thick PDMS sheet. On both PDMS rectangles, cut openings corresponding to the channels and the in-let/out-let of the device. 
		NOTE: The pre-cut openings in the PDMS rectangles are meant to prevent the channels from collapsing during pressing.</li>
		<li>Stack in the following order from the bottom: one PDMS rectangle with pre-cut openings, one PDMS disc (in contact with the bottom window, cut in step 4.3.2), the two finger-pressed halves of the device, the second PDMS disc (sitting on the top window), and lastly the second PDMS rectangle with pre-cut openings (Figure 5b).</li>
		<li>Place this assembly in the vacuum press setup such that it is sandwiched between 2 plates and seal the plastic bag (Figure 5c). Turn on the vacuum pump and evacuate the assembly. Let the vacuum pump reach its base pressure or apply the vacuum for at least 10 min. 
		NOTE: The base pressure achieved depends on the vacuum pump used and quality of the sealing of the plastic bag.</li>
		<li>Expose the evacuated assembly to UV with a broad-band Hg gas lamp at 270 W for 15 min. Turn off the vacuum pump and let the assembly slowly vent to atmospheric pressure before removing the final device from the assembly.</li>
	</ol>
	</li>
</ol>

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<li>Liu, K. Z., Xu, M., Scott, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomolecular+characterisation+of+leucocytes+by+infrared+spectroscopy%5BTitle%5D)+AND+%22Br+J+Haematol%22%5BJournal%5D)">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.sciencedirect.com/science/journal/09242031/38">IR microspectroscopy of live cells.</a> Vibrational Spectroscopy. 38 (1-2), 185-191 (2005).</li>
<li>Rahmelow, K., Hubner, W. <a target="_blank" href="https://www.osapublishing.org/as/abstract.cfm?uri=as-51-2-160">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/pubmed?term=((ATR-FTIR+spectroscopic+imaging%3A+recent+advances+and+applications+to+biological+systems%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">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/pubmed?term=((Open-channel+microfluidic+membrane+device+for+long-term+FT-IR+spectromicroscopy+of+live+adherent+cells%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">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/pubmed?term=((Microfluidic+approaches+to+synchrotron+radiation-based+Fourier+transform+infrared+%28SR-FTIR%29+spectral+microscopy+of+living+biosystems%5BTitle%5D)+AND+%22Protein+Pept+Lett%22%5BJournal%5D)">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., Pézolet, M. <a target="_blank" href="https://www.osapublishing.org/as/abstract.cfm?uri=as-43-3-538">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.sciencedirect.com/science/article/pii/S0924203110000299">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.sciencedirect.com/science/article/pii/S0924203110000184">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/pubmed?term=((Infrared+microspectroscopy+of+live+cells+in+microfluidic+devices+%28MD-IRMS%29%3A+toward+a+powerful+label-free+cell-based+assay%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">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/pubmed?term=((SU-8+bonding+protocol+for+the+fabrication+of+microfluidic+devices+dedicated+to+FTIR+microspectroscopy+of+live+cells%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">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/pubmed?term=((IR-Live%3A+fabrication+of+a+low-cost+plastic+microfluidic+device+for+infrared+spectromicroscopy+of+living+cells%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">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>
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The authors have nothing to disclose.

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.

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

soft lithographic procedure for producing plastic microfluidic devices with view-ports transparent to visible and infrared light

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 µm between the two windows. These spectra demonstrate that the fabrication approach...

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

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Learn soft lithography for fabricating plastic microfluidic devices compatible with Fourier transform infrared microspectroscopy. This cost-effective method enables label-free biochemical imaging of live cells without damaging samples.

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light (Video) | JoVE Journal

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

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