Chapter 3: 3-D Microfluidic Network Fabrication: Making Cell Culture Array on Silicon
3.2 Experimental
3.2.1 Chip Design
This chapter will discuss the development of an integrated method to fabricate monolithic 3-D microfluidic networks and the fabrication of an on-chip cell culture device with an integrated combinatorial mixer based on such technology. As a first example, the device has a combinatorial mixer with three inputs and one control channel.
The combinatorial mixer is capable of generating the eight combinatorial streams simultaneously for delivery to the eight culture chambers. Our fabrication method is based on the surface micromachining of Parylene C and can be scaled up to achieve high- throughput combinatorial cell-based assays. The packaging scheme to provide fluidic connection to the chip has been developed, and the capability to perform cell culture on this device has been demonstrated. Finally, the integrated combinatorial mixer has successfully been used to expose cells to different combinations of compounds.
3.2 Experimental
inputs into the seven possible outputs. For example, compounds A, B, and C, will be recombined into A, B, C, A+B, A+C, B+C, and A+B+C. Note that one control channel that receives an unmixed input stream is also included as the 8th output. One key feature of the chip is the overpass structure that allows one microfluidic channel to cross over other microfluidic channels, and such fluidic network component makes the combinatorial mixer possible. Solutions containing different compounds will be injected into the chip via the fluid inlets of the combinatorial mixer, and the outputted combinatorial streams will flow into the culture chambers.
Figure 3-1. Layout of the device design showing the components of the 1 cm × 1 cm chip.
The device is realized by the surface micromachining of Parylene C (poly(chloro- p-xylylene)). Several of the beneficial properties of Parylene C make it an ideal material for both 3-D microfluidic fabrication and constructing a cell culture and cell-based assay device. Parylene C is compatible with lithographic CMOS/MEMS fabrication processes and our previous work has reported that multiple layers of Parylene C can be deposited and patterned to build complicated mechanical structures. This will allow our 3-D
microfluidic device to be monolithically fabricated, favorably avoiding the multilayer bonding process. Another aspect of this device is the ability to culture cells on-chip and Parylene C is suitable for this purpose. Because Parylene C has been shown to be biocompatible and resistant to corrosive body fluids, it was used in many implantable devices [35, 36]. Long-term cell culturing using Parylene C devices have been demonstrated [81]. Also, Parylene C is chemically inert and stable toward many organic solvents [34]. In addition, Parylene C is transparent in the visible range and this will allow cells to be easily observed with light microscopy. All these valuable properties of Parylene C make it the material of choice for fabricating our device.
3.2.2 3-D Microfluidic Fabrication
The device has two levels of microfluidic channels, and its five-mask fabrication process is shown in Figure 3-2. The 3-D rendition of the process flow is also included to aid visualization. The first-level channels were initially deposited, followed by the deposition of the overpasses and culture chambers. The device was fabricated using our developed Parylene surface micromachining technology: alternating layers of Parylene C and sacrificial photoresist are deposited, and the sacrificial photoresist between two layers of Parylene defines the channel. After the photoresist is removed, the remaining hollow Parylene structures become the fluidic networks of the chip.
Figure 3-2. The monolithic fabrication process for making the three-dimensional microfluidic networks. (a) Cross sectional view of the fabrication process flow. (b) Three- dimensional rendition of the fabrication process flow. Inserts in (2) and (4) show how the overpass is made by first etching open the Parylene and joining the two etched regions with the 2nd sacrificial photoresist.
The detailed fabrication process can be broken down into the following steps.
First layer Parylene coating. Before any processing, the silicon wafer was cleaned with Piranha solution (H2SO4: H2O2, 3:1) at 120oC for 5 min. The wafer was then soaked in 0.5% silane A-174 (gamma-methacryloxy-propyltrimethoxysilane, Specialty Coating
(a)
(b)
Systems, Indianapolis, IN) solution (DI water: isopropyl alcohol: A-174, 100:100:1) for 15 minutes to promote the adhesion between the substrate and Parylene C. The Piranha treatment not only cleaned the surface, but also oxidized the substrate surface. The oxidized surface facilitated the binding of the adhesion promotion A-174 molecule onto the surface. The chemical structure of the A-174 silane coupling agent follows the general structure of silane-based coupling agents: RnSiX(4-n). R is the nonhydrolyzable organic moiety that binds to the coating polymer, which is Parylene C in our case. The X represents alkoxy moieties, and is methoxy in A-174. During the treatment, the methoxy group is hydrolyzed to methanol, forming hydroxyl group that can form hydrogen bonding with hydroxyl group on the substrate. Eventually, covalent Si-O bonding is formed in between the substrate and the coupling agent [82]. The other side of the silane molecule serves to grab onto the Parylene molecules to promote the adhesion onto the surface. After the treatment, a first thin adhesion layer of Parylene C was deposited (3 µm) to cover the entire wafer with a Cookson Electronics PDS 2010 system (Specialty Coating Systems, Indianapolis, IN). The subsequent deposited Parylene layers in the following processes are attached to this first Parylene layer instead of the silicon substrate and this is important because Parylene adhesion to silicon without proper treatment can be very weak. A-174 adhesion promoter cannot be applied for the subsequent Parylene depositions because when there are photoresist patterns on the substrate, isopropyl alcohol inside the treatment solution will quickly dissolve the photoresist.
First layer channel. The first sacrificial photoresist layer (AZ4620 from Clariant, Charlotte, NC) was spin-coated (15 µm) and patterned with photolithography to define the first-level channels. The sacrificial photoresist was hard baked at 120oC for 6 hours.
To promote adhesion of the next Parylene layer to the first Parylene layer, the surface was roughened using oxygen plasma and cleaned with 5% HF for 30 seconds. A second layer of Parylene C (10 µm) was then deposited to cover the sacrificial photoresist to form the first-level channels.
Parylene patterning. Parylene C was patterned using oxygen plasma so the areas where the overpass structures would be joined were exposed. This Parylene patterning also opened the area where the mixer and the culture chamber would be connected. To pattern the Parylene, metal mask was used because oxygen plasma can also etch photoresist at a rate comparable to Parylene etching, so a very thick photoresist would have to be used. Also, when striping the masking photoresist, the sacrificial photoresist can be partially dissolved and this can lead to debris clogging the channel or trapped air expanding and destroying the channel during subsequent thermal processes. Cr/Au (200/2500Å) layer was deposited using an e-beam evaporator. To pattern the metal, a 15 µm AZ4620 photoresist was patterned on top of the metal, and the metals were etched using Au etchant type TFA (potassium iodide (KI) + iodine (I2)) (Transene, Danvers, MA) and CR-7 Cr etchant (9% ceric ammonium nitrate + 6% perchloric acid in water) (Cynateck, Freemont, CA) . The photoresist was stripped using acetone. The Cr/Au metal layer was then used as an etch mask for Parylene patterning in oxygen plasma. The metals were stripped after Parylene patterning.
Second layer channel. A second sacrificial photoresist was spin-coated (32 µm) and patterned to define the overpass structures and the culture chambers. This second sacrificial photoresist covered the etched open areas, and the overpasses spanned several of the first-level microfluidic channels. A third layer of Parylene C (10 µm) was
deposited after the same adhesion promotion treatment procedure as described in previous steps. This Parylene was patterned using metal as mask and oxygen plasma etching.
SU8 planarization and chip release. The whole chip was strengthened and planarized with patterned 100 µm SU8 (MicroChem, Newton, MA) to facilitate the following packaging of the chip. The wafer was diced to yield chips with dimensions of 1 cm × 1 cm. Finally, the chips were soaked in 65oC IPA (isopropyl alcohol) to dissolve the sacrificial photoresist in about one week.