FABRICATION OF DOUBLE-SIDED MICROFLUIDIC STRUCTURES VIA 3D PRINTED TRANSFER MOLDING

In this work, we demonstrate the use of 3D printed molds for rapidly fabricating multi-layer PDMS-based microfluidic devices. Because 3D printing allows for versatile and cost-effective mold construction, it can produce significantly more varied features than those generated by soft lithography. We first discuss adaptations to single-layer 3D molding, including a glass bonding technique to compensate for the limitations of surface roughness and 0.55mm built-in inlet and outlet ports to eliminate fabrication steps. Next we introduce two-sided fabrication methods, facilitated by novel built-in alignment marks. These techniques allow the construction of formerly difficult to achieve features such as non-planar 350 μm membranes, used to fabricate a single-layer membrane valve which actuates at 200kPa, and single-layer microfluidic vias, used to generate 3D flow patterns. Lastly, we demonstrate an intra-layer bonding technique where a custom 3D printed stamp selectively applies liquid PDMS adhesive, compensating for surface roughness while preventing channel clogging. Together, these techniques enable the rapid assembly of multi-layer PDMS-based microfluidic devices, combining the versatility and speed of emerging 3D printing technology with the known mechanical and biological properties of PDMS favored by microfluidic researchers. INTRODUCTION Microfluidics has rapidly advanced in the fields of chemical and biological research, commonly known as Lab-on-a-Chip (LoC), since 1980s due to its unique ability to make low-cost, highthroughput platforms [1-3]. The most far-reaching breakthrough in microfluidics has been the development of soft-lithography using rigid micromachined molds to shape elastomeric polymers. Among the polymeric materials, Poly(dimethylsiloxane) (PDMS) is commonly used due to its numerous ideal properties, including its low cost, strength, transparency, and especially biocompatibility [4]. Traditional methods for fabricating microfluidic devices involve photolithography to construct micro-molds with very fine features; however, the process can be lengthy and costly. Additionally, soft lithography is limited to rectilinear features constructed through additive micromachining processes. [5] For example, while circular channels are common in large fluidic systems and are beneficial for microfluidic devices with internal movable components, [6] to date few groups have developed techniques for circular channels in microfluidics. [7]. The increasing demand for microfluidic devices is particularly high for double and multi-layered devices to allow for the implementation of more sophisticated structures and components (e.g. valves, pumps, and other active control mechanisms). Although multi-layer PDMS manufacturing techniques have been demonstrated, they are often time consuming, labor-intensive, and inaccurate. 3D printing has presented a unique route to build multi-layer microfluidic devices directly or indirectly with the additional molding process. For example, various groups have used 3D printers previously to make simple microfluidic devices with complex and truly 3D geometries, including microfluidic devices without moving elements, such as resistors, [8] mixers, modular components, [9] and microfluidic devices with movable components, such as capacitors, diodes and transistors. [10] Although 3D printed microfluidic devices are currently limited by: (1) the available resolution of the printer; (2) surface roughness; [11] and (3) material types, [12] the rapid developments of the 3D printing technologies are expected to advance and address these matters in the coming years. Additionally, while direct 3D printing is a rapid process for prototyping, for making multiple copies of microfluidic devices, it is slower, more expensive, and less reliable than transfer molding performed by pouring polymer into a mold. Thus, we present a hybrid approach to combine 3D printing with molding. This work advances the 3D printed transfer molding technique from single-sided microfluidics [13,14] to multi-layer microfluidic manufacturing, utilizing the ease of 3D printing to create multiple molds with alignment structures to shape multiple layers of PDMS structures and quickly assemble them at the end. Using this molding method, we create complex geometries in PDMS, including vias, thin membranes, and rounded channels and demonstrate rapid assembly of multi-layer microfluidic devices using built-in alignment marks which allow precise positioning of each layer without the need for a microscope. EXPERIMENTAL TECHNIQUES 3D printed molds Microfluidic devices were designed and converted from a positive to a negative mold shape using the computer-aided design program SolidWorks. 3D printing of molds was achieved using a Projet 3000 3D printer. During printing, the Projet 3000 deposited layers of structural epoxy (VisiJet® EX200 plastic material) and sacrificial wax (VisiJet® S100 support material); the wax was used as a temporary support for hollow spaces as well as to provide a foundational layer for the mold, and was removed during postprocessing. [15] Mold post-processing Following printing, the molds were cleaned to remove the sacrificial wax. First, the molds were baked in a VWR 1330 FM oven 75oC for 45 min to melt the sacrificial wax. The molds were then washed in a sequence of three cleaning baths for 10 min in each bath to remove leftover wax: warm Bayes mineral oil, Ajax dish detergent in water, and potable water. The baths were heated to 75oC to ensure the wax did not re-solidify, and were placed on a hotplate with a magnetic stir bar to enhance removal of wax, oil, and soap, respectively. The molds were then dried and residual water was removed by baking the molds at 80oC for 60 min. After cleaning and drying, the 3D printed molds were treated with an anti-adhesive agent (Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOTS), Sigma Aldrich) gas, making the surface hydrophobic to facilitate the rapid removal of PDMS. [16] The molds were placed in vacuum desiccator with 10 drops of PFOTS agent for 30 min. Shorter times resulted in PDMS bonding to the mold and longer times caused a build-up of PFOTS which inhibited complete curing of the PDMS near the surface. [11,17] PDMS molding The 3D printed molds were placed onto a foil-wrapped 3D printed molding tray, which reduces PDMS waste. PDMS (Sylgard 184 Elastomer Kit) was prepared using the standard 10:1 as the base:curing agent ratio. The PDMS mixture was degassed in a vacuum chamber for 10 minutes and then poured into the 3D printed molds. The filled molds were then placed in the vacuum chamber for 45 min to degas and increase PDMS conformity. Following the degassing, the molds were baked in an 80 oC oven for 50 min. The PDMS microfluidic devices were removed from the molds by first cutting away excess PDMS and then by manually peeling the PDMS loose from the mold. The finer the printed features, the more carefully this removal must be performed. Provided no features have broken during the de-molding process and PDMS did not permanently bond to the mold, the molds are reusable without an additional cleaning process. If during de-molding PDMS adheres excessively to the 3D printed mold, the hydrophobicity of the molds must be ‘recharged’ by repeating the PFOTS treatment (approximately every 10-20 moldings). RESULTS AND DISCUSSION Figure 1 illustrates the process flow for fabricating PDMS microfluidic devices using 3D printed molds. In this example, three microchannels with elliptical-shape reservoirs of dimensions 1.5x5.9 mm2, 1.1x5.9 mm2 and 0.7x5.9 mm2 were designed as shown in Fig. 1a. The overall dimension of the mold was 20x20x2 mm3 with quarter-circle (R = 2.5mm) pillars at the four corners facilitating removal of the PDMS after curing. Each channel was constructed with built-in fluid inlets and outlets molded from pillars 0.55mm in diameter and 5mm in height. The inlet pillars were strengthened by widening them halfway down to 1mm in diameter. Figure 1. Illustration of the 3D printed transfer molding technique and process. The microfluidic device mold is designed using CAD and the PDMS structure can be bonded onto a glass substrate to make the enclosed channels and reservoirs system. The complex multi-level microfluidic device is fabricated using the 3D printed mold shown, and includes two layers of overlapping fluid flow, an elliptical membrane, a “Quake” membrane value, multiple microfluidic vias, built-in fluid inlets, and alignment marks to create a stacked, multi-layer device. The device mold was fabricated via the 3D printing process and PDMS was applied, cured and released from the mold by means of the PDMS molding steps described in the Experimental section. Built-in fluid inlets can be easily incorporated to the device through the mold design, further simplifying fabrication by eliminating the need for a hole-punching step. These built-in inlets were reliable to pressures of 4 ATM. Narrower inlets were shown to hold against higher opposing pressures as the channel inlet gripped the couple more strongly. However, narrower inlets faced trade-offs with the fragility of the 3D printed mold, breaking during post-processing. The PDMS device was then bonded to a glass slide to create closed microfluidic channels. Clearly, this 3D printed transfer molding and bonding technique can be used to fabricate conventional microfluidic circuit devices commonly produced by the soft lithography methods with the following advantages: faster and less complex process steps; easy to create complex 3D geometries; ability to fabricate circular channel cross-sections; and built-in microfluidic connectors. For multi-layer devices, a PDMSPDMS bonding technique has been developed with a 3D printed stamp as well as the use of built-in alignment marks for rapid assembly of more than one layer of prototype PDMS structures in this work without a microscope. These techniques make possibl