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

Summary

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

Abstract

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.

Introduction

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 composition^1,2. 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 µm⁴.

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 chamber^3,8,9. 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 spacer^9,10,11,12. 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.

Protocol

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).

1. Pattern definition
   1. Spin coat a 4 inch silicon wafer with SU-8 3010 negative photoresist at 2,300 rpm for 30 s.
   2. Soft-bake the photoresist on a hot plate at 65 °C for 2 min and then at 95 °C for 8 min.
   3. 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.
   4. Remove the wafer and apply a post exposure bake at 65 °C for 1 min and then at 95 °C for 2 min.
   5. 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 µm and below. See Figure 2b for the picture of the actual silicon mold.
2. Silanization of silicon mold
   1. 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.
   2. Place the mold in a vacuum jar with 50 µL 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.

2. Preparation of PDMS Secondary Mold

1. PDMS mixing
   1. 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.
   2. 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.
2. Mold replication
   1. 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 °C for 2 h on a hot plate to cure the mixture.
   2. 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.
   3. 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).
3. Silanization of the PDMS replica (same as in step 1.2)
   1. 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.
   2. Place the mold in a vacuum jar with 50 µL of silane and leave the jar in the vacuum state (1-10 mbar) for at least 2 h.

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.

1. 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.
2. 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.
3. Mix the PDMS elastomer and curing agent 10:1; be sure to prepare a sufficient amount of PDMS to entirely submerge the templates.
4. 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.
5. 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 °C for 2 h on a leveled hot plate to cure the mixture.
6. 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.
7. 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.
8. 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.

4. Microfluidic Device Fabrication

1. Fabrication of the patterned half of the device (i.e. , with device layout)
   1. 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.
   2. 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).
   3. Take the PDMS mold made in step 2 and place a thin UV-transparent plate (in this case, a quartz plate, 500 µm 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.
   4. 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).
   5. 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 µm) between the PDMS mold and CaF₂ window (Figure 4e-4f).
   6. 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.
   7. 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.
2. Fabrication of the flat half of the device (i.e. , without device layout)
   1. 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.
   2. 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.
   3. 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.
   4. Gradually dispense drops of NOA at the in-let of the PDMS template and let it slowly fill the cavity.
   5. 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.
   6. Peel off the PDMS sheet from the top of the NOA layer and carefully remove the cured NOA layer from the PDMS template.
3. Bonding of the two halves of the device
   1. 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.
   2. Cut two circular discs out of a 1 mm thick PDMS sheet using an 8 mm diameter puncher (Figure 5a).
   3. 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.
   4. 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).
   5. 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.
   6. 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.

Results

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...

Discussion

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 µm 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 ...

Materials

List of materials used in this article

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