Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices

Summary

This project allows small laboratories to develop an easy-to-use platform for the fabrication of precise multilayer microfluidic devices. The platform consists of a three-dimensionally printed microscope mask alignment adapter using which multilayer microfluidic devices with alignment errors of <10 µm were achieved.

Abstract

This project aims to develop an easy-to-use and cost-effective platform for the fabrication of precise, multilayer microfluidic devices, which typically can only be achieved using costly equipment in a clean room setting. The key part of the platform is a three dimensionally (3D) printed microscope mask alignment adapter (MMAA) compatible with regular optical microscopes and ultraviolet (UV) light exposure systems. The overall process of creating the device has been vastly simplified because of the work done to optimize the device design. The process entails finding the proper dimensions for the equipment available in the laboratory and 3D-printing the MMAA with the optimized specifications. Experimental results show that the optimized MMAA designed and manufactured by 3D printing performs well with a common microscope and light exposure system. Using a master mold prepared by the 3D-printed MMAA, the resulting microfluidic devices with multilayered structures contain alignment errors of <10 µm, which is sufficient for common microchips. Although human error through transportation of the device to the UV light exposure system can cause larger fabrication errors, the minimal errors achieved in this study are attainable with practice and care. Furthermore, the MMAA can be customized to fit any microscope and UV exposure system by making changes to the modeling file in the 3D printing system. This project provides smaller laboratories with a useful research tool as it only requires the use of equipment that is typically already available to laboratories that produce and use microfluidic devices. The following detailed protocol outlines the design and 3D printing process for the MMAA. In addition, the steps for procuring a multilayer master mold using the MMAA and producing poly(dimethylsiloxane) (PDMS) microfluidic chips is also described herein.

Introduction

A well-developed and promising field in engineering research is microfabrication because of the vast expanse of applications employing microfluidic platforms. Microfabrication is a process wherein structures are produced with µm- or smaller-sized features using different chemical compounds. As microfluidic research has developed over the last 30 years, soft lithography has become the most popular microfabrication technique with which to produce microchips made from poly(dimethylsiloxane) (PDMS) or similar substances. These microchips have been widely used for the miniaturization of common laboratory practices^1,2,3,4 and have become powerful research tools for engineers to mimic reaction processes^5,6,7, study reaction mechanisms, and mimic organs found in the human body in vitro (e.g., organ-on-a-chip)^8,9,10. However, as the complexity of the application increases, it is typical that a more complex microfluidic device design allows for better replication of the real-life system it is intended to imitate.

The basic soft lithography procedure involves coating a substrate with a photoresist substance and placing a photomask over the coated substrate before subjecting the substrate to UV light¹¹. The photomask has transparent regions that mimic the desired pattern of the microfluidic device channels. When subjecting the coated substrate to UV light, the transparent regions allow the UV light to penetrate through the photomask, causing the photoresist to be crosslinked. After the exposure step, the un-crosslinked photoresist is washed away using a developer, leaving solid structures with the intended pattern. As the complexity of the microfluidic devices becomes greater, they require multiple-layer construction with extremely precise dimensions. The process of multilayer microfabrication is much more difficult compared to single-layer microfabrication.

Multilayer microfabrication requires precise alignment of the first layer features with the designs on the second mask. Normally, this process is performed using a commercial mask aligner, which is expensive and requires training to operate the machinery. Thus, the process of multilayer microfabrication is typically unattainable for smaller laboratories that lack the funds or time for such endeavors. While several other custom-built mask aligners have been developed, these systems often require the purchase and assembly of many different parts and can still be quite complex^12,13,14. This is not only expensive for smaller laboratories, but also requires time and training to build, understand, and use the system. The mask aligner detailed in this paper sought to alleviate these issues as there is no need for the purchase of additional equipment, only requiring equipment that is typically already present in laboratories that produce and use microfluidic devices. In addition, the mask aligner is fabricated by 3D printing, which with the recent advancement of 3D printing technology, has become readily available to most laboratories and universities at an affordable cost.

The protocol detailed in this paper aims to create a cost-effective and easy-operation alternative mask aligner. The mask aligner detailed herein can make multilayer microfabrication feasible for research laboratories without conventional fabrication facilities. Using the microscope mask alignment adapter (MMAA), functional microchips with complex features can be achieved using a regular UV light source, optical microscope, and common laboratory equipment. The results show that the MMAA performs well with an example system using an upright microscope and a UV light-exposure box. The MMAA produced using the 3D printing process was used to acquire a bilayer master mold of a herringbone microfluidic device with minimal alignment errors. Using the master mold fabricated with a 3D-printed MMAA, microfluidic devices were prepared with multilayered structures containing alignment errors of <10 µm. The alignment error of <10 µm is minimal enough to not hinder the application of the microfluidic device.

In addition, the successful alignment of a four-layer master mold produced using the MMAA was confirmed, and alignment errors were determined to be <10 µm. The functionality of the microfluidic device and minimal alignment errors validate the successful application of the MMAA in creating multilayer microfluidic devices. The MMAA can be customized to fit any microscope and UV exposure system by making minor changes to the file in the 3D printer. The following protocol outlines the steps necessary to fine-tune the MMAA to fit the equipment available in each laboratory and 3D-print the MMAA with the required specifications. In addition, the protocol details how to develop a multilayer master mold using the system and subsequently produce PDMS microfluidic devices using the master mold. Generation of the master mold and microfluidic chips then allows the user to test the effectiveness of the system.

Protocol

1. Designing the MMAA

1. Obtain the dimensions of the tray of the available UV light emission system to be the upper bound for the dimensions of the wafer holder (or UV exposure unit) shown in Figure 1. As shown in Figure 2A, measure the diameter (d) of the inner circular rim, the inner height (h) of the UV light emission system's tray, the total width (w), and length (l) of the tray.
   NOTE: As an example, the available UV light exposure system had inner tray dimensions of 5 inch (") x 5" x 0.25" with a 4" circular cut-out. The dimensions of the MMAA were then designed to be no greater than the inner tray dimensions to fit properly and sit flat within the tray of the system as shown in Figure 2B. See Figure 3 for the 3D-printed pieces of the MMAA: photoresist-coated silicon wafer and a fastener to fix the setup to the microscope.
2. Measure the length between the screws on the available upright microscope stage that hold the slide holder in place. Additionally, measure the width of the screws. Apply these dimensions to customize the magnetic holder (Figure 1) to fit the available microscope to allow for easy and precise fixation of the MMAA to the microscope (Figure 4A).
3. Using an available computer design application, customize the wafer holder and magnetic microscope fastener to fit within the measured dimensions. Design the height, width, and length of the wafer holder to be no greater than the height (h), width (w), and length (l) of the UV light emission system's tray. In addition, include the circular cut-out at the bottom of the wafer holder with the same diameter (d) as the UV light emission system's tray. Generate STL or CAD files for the two pieces of the MMAA to be used for 3D printing of the device (see Supplemental Material).

2. 3D Printing the MMAA

1. Upload the generated STL or CAD files to the available 3D-printing software. 3D-Print the two pieces of the MMAA by following the appropriate procedure for the 3D process and printer used. Complete the pieces by following any required post-printing steps (e.g., removal of support material, removal of uncured resin, additional washing or curing steps). Alternatively, use an available 3D printing facility to have the designed pieces printed and completed elsewhere.
2. Ensure the wafer holder fits well and sits flat inside the tray of the available UV light exposure system (Figure 2B). Additionally, ensure that the microscope fastener is attached to the microscope stage and can be moved easily using the knobs that control the x- and y- positions of the microscope stage (Figure 4A).
3. Once the pieces have been finalized, insert and fix the magnets into the wafer holder and microscope fastener (Figure 3A), using super glue or any other fixing substance. Allow for the glue to dry before testing the system.
   ​NOTE: If desired, a protype piece can first be printed using a Fused Deposition Modeling (FDM) 3D printer to save resources and money¹⁵. This protype can then be assessed for accurate fit to the available equipment, and the design can then be modified, if needed. The final device can then be printed using a more accurate process (e.g., Stereolithography) for better precision. The final device can also be printed with a translucent finish for optimal use under the microscope.

3. Experimental testing of the MMAA

1. Design and printing of the microfluidic device photomasks with alignment markers
   1. Use a computer design application to design photomasks for the desired bilayer microfluidic device.
   2. Include additional structures on the side of the microfluidic device channel structures that will act as alignment markers (closer towards the edge of the photomask/master mold) as shown in Figure 5A,B. Ensure there is one alignment marker on each side of the microfluidic device (for a total of at least four). In addition, ensure the photomask contains a straight edge that can align perfectly with the straight edge of the silicon wafer.
      NOTE: The higher intricacy of the alignment marker structure will allow for greater alignment accuracy of the additional layers. At the least, a simple cross structure with measurements of 1 mm x 1 mm should be used (Figure 6A). An example of the alignment markers can be seen in the corners and bottom middle edge of Figure 5A,B, which depict the first- and second-layer photomasks used to generate a double-layer master mold.
   3. Print the photomasks either through a commercial vendor or through other accessible facilities
2. Creation of the bilayer master mold using the MMAA (photolithography)
   1. Using standard photolithography techniques and the photoresist manufacturer's instructions, create the first layer of the master mold using the first layer photomask¹⁶. Use a 4" silicon wafer with the appropriate photoresist (i.e., SU-8) to create the desired layer thickness. Ensure the first layer thickness is greater than the subsequent layers for easy identification of the alignment markers.
   2. Use a light-colored marker pen (e.g., gold) to color the first layer's alignment markers on all four sides.
   3. Using the photoresist manufacturer's instructions, initiate the second layer of the master mold by spin-coating the photoresist onto the wafer and performing the soft bake¹⁶. Insert the coated wafer into the wafer holder of the MMAA (Figure 3B) and fix the coated wafer to the MMAA using tape.
   4. Attach the wafer holder to the available upright microscope using the magnetic microscope fastener (Figure 4A). Move the position of the MMAA using the x- and y-direction knobs of the microscope stage until one of the colored alignment markers on the wafer is in view through the microscope lens.
   5. Insert the second-layer photomask into the wafer holder, on top of the coated wafer (Figure 3C). Ensure that the first-layer's colored alignment markers can be partially seen through the alignment markers on the photomask. 
   6. Attach the photomask to a scissor lift (also known as a support jack) through one of the side cut-outs (Figure 4B) with tape. Use the scissor lift to adjust the z-direction position of the photomask until it lies right above the coated wafer (Figure 3C).
      ​NOTE: The scissor lift allows for fine adjustment of the z-position of the photomask, as the scissor lift can be used to move the position of the attached photomask in the z-direction.
   7. While keeping the photomask still, look through the microscope lens and identify the first-layer’s colored alignment markers beneath the alignment markers of the photomask. Use the x- and y-direction knobs of the microscope stage to move the position of the MMAA (Figure 4D). Adjust the position of the MMAA until the alignment marker on the photomask is superimposed with the colored alignment marker on the first layer (Figure 6A,B) by observing the position of the alignment markers through the microscope lens.
   8. Carefully apply a slight force to the photomask and use tape to secure the photomask in place on top of the coated wafer. Detach the photomask from the scissor lift. Ensure all four alignment markers on the photomask are in alignment with the four alignment markers on the first layer.
   9. Once the alignment is achieved, carefully detach the wafer holder from the microscope stage. Insert the glass top plate on top of the wafer and photomask to decrease the gap between the two pieces (Figure 1). Place the entire wafer holder into the available UV light exposure system as shown in Figure 4E. Expose the second layer for the appropriate time and light intensity as described in the photoresist manufacturer's instructions¹⁶.
   10. Remove the wafer holder from the UV light exposure system. Remove the coated wafer from the wafer holder and detach the photomask from the wafer. Complete the processing of the second layer (e.g., post-bake, developing, and rinse and dry) as per the photoresist manufacturer's instructions¹⁶.
       ​NOTE: The exact spin-coating, soft baking, exposing, post-baking, and developing conditions (time, temperature) will vary based on the photoresist being used and the desired layer thickness. The actual conditions and exact photolithography procedure should be based on the photoresist manufacturer's instructions.
3. Preparation of a microfluidic device using the master mold (soft lithography)
   1. Retrieve the master mold and secure it in the middle of a 150 mm x 15 mm plastic Petri dish with tape.
   2. Prepare ~15-20 g of PDMS based on the manufacturer's instructions. Place the PDMS in a vacuum chamber or let it rest until free of any bubbles. Pour the PDMS into the Petri dish containing the master mold.
   3. Let the Petri dish with the master mold rest on the countertop until the PDMS is free of any bubbles. Place the Petri dish in an oven at 65 °C until the PDMS is fully cured (at least 3 h).
   4. Cut out the PDMS to reveal the microchannel structures. Cut the PDMS around the microchannel structures into separate microchips and create the inlet and outlet holes for the microfluidic device. Use tape to gently remove any small particulates that may lie on the PDMS surface.
   5. Complete the microchip fabrication by bonding the PDMS chip to the PDMS or a microscope slide by plasma-treating the PDMS chip and the additional substrate.
4. Determination of the alignment error
   1. Retrieve the master mold and use the upright microscope to determine the gap distance (alignment error) between the first layer and second layer. Do this by simply measuring the distance by which the second layer is shifted and misaligned from the first layer on the microchannel structures (see Figure 5D for an example of a measured gap distance).
   2. Use the upright microscope to determine whether the PDMS chip contains channel walls that are straight with clear device edges. Additionally, check the PDMS chip for any possible defects that may hinder device functionality.
      NOTE: The master mold fabrication (sections 3.2 and 3.3) may need to be repeated to achieve a lower alignment error. Repeated practice using the MMAA is shown to enhance the user's ability to create a well-aligned master mold. In addition, images can be obtained by scanning electron microscopy (SEM) (Figure 7) to confirm the alignment error.

Results

Through the optimization and use of the MMAA (Figure 1), multilayer master molds with minimal alignment error were fabricated. The final MMAA was fabricated using the fused filament fabrication (FFF) 3D-printing process (Figure 2). The FFF process confers increased accuracy for the desired device dimensions. The MMAA consists of two main pieces (Figure 3): the base piece and the custom fastener. The base piece consists of the U...

Discussion

The aforementioned protocol outlines the procedure for 3D-printing an MMAA and using the system to create a precise, multilayer, microfluidic device master mold. Although the device is easy to use, there are critical steps within the protocol that require practice and care to ensure proper alignment of the master mold layers. The first critical step is the design of the MMAA. It is essential when designing the MMAA to determine the exact measurements for the device that will allow for a proper fit inside the UV light exp...

Materials

Name	Company	Catalog Number	Comments
Acrylonitrile Butadiene Styrene (ABS), 3D Printing Filament			Provided by the Texas Tech University 3D printing facility
BX53, Upright Microscope	Olympus
Form 2, Stereolithography 3D printer	Formlabs
Advanced Hot Plate Stirrer	VWR	97042-642
Isoproyl Alcohol, 70% (v/v)	VWR	BDH7999-4
Light Colored Marker	Sharpie
Magnets, 3 mm x 3 mm	WOTOY		ASIN #: B075PLVW8W
SYLGARD 184 Silicone Elastomer Kit	DOW	4019862
Petri Dish, 150 mm x 15 mm	VWR	25384-326
Printed Photomasks	CAD/Art Services, Inc.
Aluminum Support Jack - 8" x 8", Scissor Lift	VWR	12620-904
Silicon Wafer	University Wafer	452
Sodium Hydroxide	VWR
Sonication Bath	Branson	CPX3800H
Spin Coater	Laurell Technologies Corporation	Model WS-650MZ-23NPPB
STRATASYS SR-30	MakerBot Industries, LLC	SR-30	Dissolvable support material for 3D printing
Stratasys uPrint SE 3D Printer	Computer Aided Technology, LLC
SU-8 50	Kayaku	Y131269 0500L1GL
SU-8 100	Kayaku	Y131273 0500L1GL
SU-8 Developer	Kayaku	Y020100 4000L1PE
Super glue	Gorilla Glue
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane	Sigma-Aldrich	448931-10G
Tape	Scotch
Form Cure, UV Curing Chamber	Formlabs	FH-CU-01
UV-KUB2, UV Light-Exposure Box	Kloe	UV-KUB2