Art-on-a-Chip: Preserving Microfluidic Chips for Visualisation and Permanent Display
Rebecca Soffe,*a Albert J. Mach,b Sevgi Onal,a Volker Nock,a Luke P. Lee,*c,d,e and J. Tanner Nevillf
a Department of Electrical and Computer Engineering, University of Canterbury, Christchurch, New Zealand.
b.BD Biosciences, 2222 Qume Drive, San Jose, CA 95131, USA.
c Department of Bioengineering, University of California Berkeley, CA 94720, USA.
d Berkeley Sensor and Actuator Centre, University of California Berkeley, CA 94720, USA.
e Department of Electrical Engineering and Computer Science, University of California Berkeley, CA 94720, USA.
f Berkeley Lights, 5858 Horton St, Suite 320 Emeryville, CA 94608, USA.
* [email protected], [email protected]
Abstract
“After a certain high level of technical skill is achieved, science and art tend to coalesce in aesthetics, plasticity, and form. The greatest scientists are always artists as well.” Albert Einstein. Currently, photographic images bridge the gap between microfluidic/lab-on-a-chip devices and art. However, the microfluidic chip itself should be a form of art. Here we present novel vibrant epoxy dyes in combination with a simple process to fill and preserve microfluidic chips, to produce Microfluidic Art or Art-on-a- Chip. In addition, this process can be used to produce epoxy dye patterned substrates that preserve the geometry of the microfluidic channels – height within 10% of the mold master. This simple approach for preserving microfluidic chips with vibrant, colourful, long-lasting epoxy dyes creates microfluidic chips that can be easily visualised and photographed repeatedly - for at least eleven years. Hence enabling researchers to showcase their microfluidic chips to potential graduate students, investors, and collaborators.
1. Introduction
To truly communicate the marvels around us, we cannot solely rely on the use of words. Which is powerfully demonstrated by the illustrations of Da Vinci’s inventions or the base-pair models of the DNA double helix by James Watson, Francis Crick, Maurice Wilkins, and Rosalind Franklin.[1-3] These illustrations simultaneously demonstrate the complexity and simplicity of scientific knowledge.
Furthermore, illustrations continually educate, excite, and inspire the inner scientist in us all.
Communication of complex scientific phenomena becomes more challenging when one is unable to see the marvel with one’s naked eye. For example, we can readily see water phenomena and bodies of water on the macro-scale, such as Lake Matheson in New Zealand, which was formed through glacial retreat.
Conversely, microfluidic devices are typically large enough to identify key micro-scale features with the naked eye, but an optical microscope is often necessary to fully appreciate the intricate network of microchannels. In addition, in some instances, the similarity of refractive indices of polydimethylsiloxane (PDMS) and air can play tricks on the untrained eye, which makes structural details in microfluidic chips more difficult to visualise (Figure 1).
Over the years, the focus in microfluidics has shifted from understanding and characterising fluids at the submillimetre length, to leveraging microfluidics to produce solutions for applications.[4] For example investigating physiological phenomena, such as mechanotransduction, to the development of organ-on-a- chip for drug discovery or leaf-on-a-chip for phyllosphere microbiology.[5-15] Even with a change in focus, the physical microfluidic chips used in research continue to play an important role in developing new research collaborations, enticing students and funding, and communicating research to the wider community. Therefore, researchers will often take photographs of microfluidic chips filled with food dye or produce two- or three- dimensional schematics to aid in the visualisation of the microchannels and chip function. For example, microfluidic chip images have appeared on journal covers over the years to spark wider research interest, for example to name a few: a photograph of our integrated microfluidic cell culture and lysis on a chip appeared on the cover of Lab on a Chip in December 2007;[7] a microfluidic affinity analysis device with > 7,000 valves graced the cover of Nature Biotechnology in September 2008;[16] and a schematic of a drug dissolution chip appeared on the cover of Small in September 2011.[17]
The traditional approach to photograph a microfluidic chip commonly involves a simple procedure employing food dye, and a pressure or hand driven syringe. This approach requires a permanently bonded PDMS chip and will frequently result in the incomplete filling of complex chips, or the development of microbubbles around small features. However, this can be addressed by utilizing passive filling, where the PDMS chip is vacuumed, removed from the vacuum desiccator, and droplets of food dye are placed on the inlets of the microchannels, or the chip is completely submerged in the food dye.[18, 19] Although this approach is sufficient for taking photographs for publication, it is unsuitable for showcasing physical microfluidic chips for permanent displays, presentations, or media communications. As food dye readily
evaporates overnight, leaving microfluidic chips that are only partially filled, which substantially decreases its visual aesthetics. Furthermore, if uniform drying can be achieved the vibrancy of the food colour will quickly degrade, especially when stored in ambient light or in sunlight in particular. As a consequence, these properties hinder the use of food dye to produce effective and vibrant microfluidic chips, which leave a lasting impression. To overcome these limitations, we have developed novel colourful epoxy dyes that can be used to preserve microfluidic chips with vibrant colours for display.
Here we present the process to produce microfluidic art or art-on-a-chip, through preserving microfluidic chips by using our novel coloured UV curable polymers (epoxy dyes). We use Sudan dyes to colour UV curable polymers to produce epoxy dyes with vibrant colours, which preserve microfluidic chips that can retain their vibrancy for at least eleven years. Furthermore, we demonstrate how this process can be used to pattern intricate microchannel designs onto glass and silicon substrates.
Figure 1. Empty microfluidic chip, demonstrating the difficulty of visualizing fine microchannel detail without the addition of coloured dye.
2. Results and Discussion
Impactful visualization and preservation of microfluidic chips/lab-on-a-chip devices requires the complete filling of microchannels with vibrant colours – without the presence of microbubbles. Ideally, the colours used for filling the microchannels should not degrade over time, in regard to the fidelity of the complete filling of the microchannels and colour vibrancy. In the following subsections we present results and discuss the novel epoxy dye preparation (Figure 2a-d) and microfluidic chip preservation process (Figure 2e-g). For material details and detailed methods for epoxy dye preparation and the preserving microfluidic chip process refer to the Experimental section.
Figure. 2. Epoxy Dye Preparation and Preserving Microfluidic Chip Process. To prepare the epoxy dye (a) add Sudan dye and toluene into a glass vial, and (b) thoroughly mix together. (c) Then add the desired norland optical adhesive (NOA) and thoroughly mix together. (d) Finally, evaporate the toluene in a fume hood to produce the epoxy dye. To preserve the microfluidic chips, (e) the PDMS was vacuumed and then loaded with epoxy dye using a pipette. (f) Once completely filled the epoxy dye was cured under UV light. (g) For temporary bonded chips the PDMS was peeled off to reveal an epoxy dye patterned substrate.
2.1 Preparation of the Epoxy Dye.
Initially the Sudan dyes were mixed directly with NOA 72; however, this resulted in heterogeneous epoxy dyes that were not suitable for preserving microfluidic chips. Therefore, a solvent was sought that would effectively dissolve both the Sudan dyes and NOA. Sudan dyes, when used as biological stains are typically dissolved in solvents such as isopropanol, methanol, acetone, toluene or chloroform.[20] As an initial test, we dissolved 2 mg of Sudan II (orange) in the aforementioned solvents and found that acetone, toluene, and chloroform effectively dissolved Sudan II (Figure S2). In addition, chloroform
instantaneously dissolved the NOA, and with thorough mixing, acetone and toluene were also able to dissolve the NOA. Although toluene took longer to evaporate from the small glass vials, the resulting epoxy dyes maintained their vibrant colours. This was not the case for epoxy dyes produced using chloroform or acetone, which faded during solvent evaporation and UV curing, respectively.
Norland products offers different UV curable adhesives, that are suitable to produce the epoxy dye. We found that NOA 72, which has a viscosity of 155 cps was suitable for our microfluidic chips, including our more intricate designs (Figure 3). Changing the amount of Sudan dye can produce colours with different intensity, and mixing different Sudan dyes can produce new colours, such as purple (Figure 3).
Figure 3. Preserving Microfluidic Chips with Epoxy Dye. (a) Photographs of a lab-on-a-chip platform designed for cell culture and lysis taken two weeks after being filled with (i) food and (ii) epoxy dye. The food dye filled microfluidic chip dried out and lost its vibrancy, whilst the epoxy dye filled microfluidic chip maintained its vibrant colour. (b) Microfluidic Art or Art- on-a-Chip as displayed by the beauty of the Golden Gate Bridge furnished in a wooden frame, and sections showing the (ii) inlet channels and a (iii) section of the Golden Gate Bridge. (c) Ivy nervature two-toned microfluidic chip achieved with sequential epoxy dye filling.
2.2 Curing the Epoxy Dye.
After filling the microfluidic chip with epoxy dye through passive filling, the epoxy dye was cured using a high powered UV curing system. For example, the OmniCure® S2000 Spot UV Curing System has an output power of 0.2 to 40 W/cm2. A high power system is important as PDMS is permeable to small molecules,[21] so any incomplete curing of the epoxy dye resulted in the PDMS being permanently coloured beyond the channel cross-section (Figure S3). This phenomenon was more prevalent when using a low power UV curing system, for instance a Karl Süss MA-6 mask aligner (< 10 mW/cm2, λ = 365 nm).
Extending the UV curing duration resulted in the microfluidic chip becoming warm due to the energy deposited, which often caused instantaneous dye diffusion into the PDMS (Figure S3). Alternatively applying a multi-exposure process (e.g. 540 s exposure, 60 s wait) reduced the diffusion due to thermal load, but the extended duration overall still enabled diffusion to occur. Although pretty from an artistic point of view, this was undesirable for the preservation of the microfluidic chips; as it made the microchannels difficult to fully appreciate by the naked eye. We found that the curing duration was dependent: on the thickness of the PDMS (4 mm thick PDMS attenuated the UV light by 2 mW/cm2);
microchannel geometries; optical adhesive (NOA 72 requires 5 J/cm2); and the UV curing system (output power of 0.2 to 40 W/cm2 for the OmniCure S2000), including the distance between the light source and sample (note if the light source is too close the colours can fade). Hence, the curing duration must be determined for each microfluidic chip for effective preservation (Figure 3). We also found that it was best to slightly extend the curing duration, once the minimal curing duration was determined. This was undertaken to account for any slight variances in parameters, such as the thickness of the PDMS or positioning of the microfluidic chip in reference to the UV curing system. Furthermore, it is advisable to remove any epoxy dye in the inlet holes of the microfluidic channels prior to curing. The increased thickness of the epoxy dye will increase the occurrence of incomplete curing and result in dye diffusion around the inlet holes.
2.3 Dye Comparison.
Filling a microfluidic chip with food dye using traditional processes comes with a major caveat. A microfluidic chip can only be illuminated for a short duration due to the evaporation of food dye – which is significantly noticeable after 24 h. Although this might be sufficient to acquire a photograph for journal publications or media communications, it is insufficient to permanently preserve a microfluidic chip.
Conversely, filling a microfluidic chip with epoxy dye using the preserving microfluidic chip process presented in this publication, produced a permanently illuminated microfluidic chip. To highlight the proficiency of the epoxy dyes, a comparison experiment was undertaken between the food and epoxy dyes, using a lab-on-a-chip platform designed for cell culture and lysis (Figure 3a). This particular microfluidic chip contains six chambers, with each chamber containing four trapping structures with a height of 2 µm, whilst the remaining microchannels have a height of 40 µm.[7] After two weeks of storage in a typical laboratory environment, the food dye had evaporated, whilst the epoxy dye maintained its vibrant colour.
In fact, experiments conducted over an extended duration indicated that the epoxy dyes were able to maintain their vibrancy over significant periods. For example, the microfluidic Golden Gate Bridge (Figure 3b) is still maintaining its vibrancy after eleven years of display in an office (Figure S4). We conjectured that the stability of the epoxy dyes was due to the chemical compatibility between the Sudan dyes and the optimised composition of the UV curable epoxy used – NOA 72. In addition, to the selection of a suitable solvent (Figure S2), toluene, which was able to dissolve both the Sudan dye and UV curable epoxy, to produce an epoxy dye that maintained vibrancy throughout the epoxy dye preparation and microfluidic chip preservation process.
The capacity of the epoxy dye to reveal sections of a microfluidic chip that are usually hidden from the naked eye, can be seen by admiring the microfluidic Golden Gate Bridge details close-up (Figure 3b).
Usually hidden from the naked eye, the inlet channels branching from the edges of the microfluidic Golden Gate Bridge (Figure 3b(ii)), that are loaded with coloured epoxy dye to fill specific regions (Figure 3b(iii)) are now visible. In addition, the epoxy dyes can be loaded into the microfluidic channels with minimal mixing, mainly due to low Reynolds numbers, to produce two-toned microfluidic chips through sequential dye filling. This was demonstrated in a powerful manner by filling an ivy leaf nervature chip first with orange, and then with purple epoxy dye (Figure 3c). A nervature chip has potential applications in generating fractal vascular-branching patterns for perfusable tissue constructs, patterning endothelial cells, and investigating the influence of cell behaviour (e.g. cell signalling) due to microenvironmental factors.[6,
19, 22] Hence, following the process presented here researchers are able to turn their microfluidic chips into microfluidic art or art-on-a-chip, that can be enjoyed for years to come.
2.4 Epoxy Dye Substrate Patterning.
The preserving microfluidic chip process can also be utilised to pattern substrates with microchannel designs (Figure 4 and Figure 5). To demonstrate this, we used temporary bonded microfluidic chips and peeled the PDMS away from the substrate after UV curing the epoxy dye, to reveal the patterned substrate.
The same lab-on-a-chip design that was permanently preserved in Figure 3a, was also used to pattern a glass substrate (microscope slide) by temporary bonding the PDMS to the glass and subsequently removing the PDMS (Figure 4a). This highlights the potential of patterning UV curable polymers with microstructures defined by microchannels onto substrates for biological applications or replica molding techniques.[23, 24] For example, proteins have been patterned on glass substrates, in a manner similar to microstamping, for cell-based studies or even multilayer patterning of proteins.[25-28]
For more intricate microfluidic designs, such as leaf nervature we initially had difficulty patterning the glass substrate; as the epoxy dyes would remain in the PDMS microfluidic channels when peeled away from a glass substrate (Figure 4b). We attributed that this was due to the finer details and more random spatial arrangement of the microchannels in the leaf example; which both increase the occurrence of the epoxy dye not releasing from the microchannels. Therefore, we varied different parameters of the protocol to adapt it for more intricate microfluidic chip designs. This included applying different glass cleaning processes and UV curing conditions, to changing the thickness of the PDMS. However, the epoxy dyes still remained in the microchannels. In consequence, we concluded that the adhesive force would need to be reduced, to facilitate the successful release of the epoxy dyes. This problem is similar to what has been encountered in removing PDMS from silicon, especially with high aspect ratio microstructures, and double casting of PDMS, where an anti-adhesion layer needs to be applied prior to casting. A common processes for producing an anti-adhesion layer is trichloro(1H,1H,2H,2H-perfluorooctyl)silane treatment to the mold master.[12, 29] Briefly, we oxygen plasma treated the PDMS microchannels, followed by trichloro(1H,1H,2H,2H-perfluorooctyl)silane treatment for at least an hour under vacuum. Just prior to temporary bonding the microfluidic chips, we removed the substrates from the oven and left them to cool to room temperature. We then followed the rest of the preserving microfluidic chip protocol to fill the microfluidic chips and cure the epoxy dyes. Once the epoxy dyes were cured, we peeled away the PDMS to reveal the intricate microchannel patterned substrates. The success of this adapted protocol for intricate microchannel designs, can be observed in Figure 4c with a two-toned patterned Māhoe leaf nervature on glass. The insert shows the microfluidic channels filled with blue epoxy dye along the midrib at the base of the leaf; thus, indicating that the epoxy dye reaches the corners of the microchannels. In addition to glass substrates, this adapted protocol can be utilized to pattern intricate designs onto silicon substrates.
We used this adapted protocol to pattern a silver fern and a microfluidic text example onto a silicon wafer (Figure 4d).
Although the silane treatment improves the releasing of the cured epoxy dyes, it hinders the filling of the microchannels with the liquid epoxy dye. However, this can be improved by increasing the microchannel height, whilst ensuring to consider the aspect ratios of the microfeatures. For example, the microfluidic text would not completely fill with temporary bonded, silane treated microchannels with a height of 50 µm. Nonetheless, the microchannels would completely fill in 30 to 45 minutes when the height was increased to 100 µm (Figure 4d, Figure S5) – with four-millimetre thick PDMS. In comparison, for permanently bonded chips with the same design (using the initial process) we were able to fill the microchannels of a height of 50 µm with epoxy dye in around 30 minutes.
The PDMS also had to be peeled off the substrate shortly after UV curing the epoxy dye, to enable the complete release of the epoxy dye from the microchannels. Attempts to peel away the PDMS (with microfluidic nervature) from glass the day after UV curing the epoxy dye were not successful. This occurred even with silane treated PDMS, which we conjecture was due to the cross-reactivity between the epoxy dye and the silane surface treatment.
In addition to filling microfluidic channels either permanently preserve the microfluidic chip or to pattern substrates, a substrate can be patterned with the epoxy dye using standard photolithography techniques with a variety of designs (Supplementary Information S6). However, using standard lithography comes with several limitations: (1) Approximately 750 µL of epoxy dye was required to sufficiently cover a 3”
wafer by spin coating, which is considerably more than the 50 to 100 µL required to fill a microfluidic chip. (2) The thickness of the epoxy dye is restricted by spinning speed and viscosity of the epoxy dye, whereas, a microfluidic channel can have a height of a couple of micrometres to several hundred micrometres. (3) The achievable resolution is limited as a spacer (minimum 60 µm) must be placed between the mask plate and the substrate to ensure that the epoxy dye does not cure on mask plate – which would result in the substrate being bonded to the mask plate. (4) The exposure duration was longer using a mask aligner as the UV light source compared to a Spot UV curing system (e.g. OmniCure® S2000 or Novacure), due to the weaker light source. These limitations highlight the flexibility of the microfluidic chip preserving process presented in this paper, which requires small epoxy dye volumes, offers variability in epoxy dye height, shorter curing durations, cleaner fabrication, and the ability to readily fill a microfluidic chip for communication purposes.
Figure 4. Patterned Epoxy Dye on a Substrate. (a) Epoxy patterned onto a glass substrate highlighting the potential to produce scaffolds for biological applications. (b) Epoxy dye remaining in the PDMS microchannels of an Ivy leaf nervature chip. (c) Māhoe leaf nervature patterned on a glass substrate, with an insert showing a cross-section of blue epoxy dye in the microchannel. Scale bar 250 µm. (d) Silver fern nervature and microfluidic text patterned onto a silicon wafer.
To investigate how effective the preserving microfluidic chip process is at patterning microchannels onto a substrate, we obtained comparative 3D optical profiles for the silver fern nervature and microfluidic text (Figure 5). We compared the SUEX® K100 negative tone photoresist mold master (Figure 5a), and subsequently patterned microchannels on glass (Figure 5b) and silicon (Figure 5c) substrates. The four- millimetre squared inserts highlight in further detail that the patterned structures are representative of the microchannels used to pattern the epoxy dye. Which is agreeance with the cross-section image presented in Figure 4c; which provided visual confirmation that the epoxy dye entirely fills the microchannel cross- section. The 3D optical profile comparison reveals that the resulting height of the patterned microchannels is comparable to that of the mold master, with the height of the patterned epoxy dye on both silicon and glass was within 10 µm of the height of the mold master (Figure 5, Figure S8). The slight variance of dimensions of the epoxy dye was expected due to slight shrinking of the PDMS microchannels during curing on the mold master, and the curing of the epoxy dye within the microchannels. Investigating the roughness further by using atomic force microscopy on a patterned epoxy dye leaf nervature on glass indicated that the surface had a rms roughness of 5.17 nm (Supplementary Information S7). Therefore, indicating that the pattered epoxy dye is representative of the PDMS microchannels and negligible surface roughness due to the properties of the epoxy dye.
Figure 5. 3D Optical Profiles. Optical profiles of: (a) SUEXTM K100 negative tone photoresist mold master on a silicon wafer;
(b) epoxy dye patterned on a glass substrate; and (c) epoxy dye patterned on a silicon substrate. Each (i) large area scan has a corresponding (ii) small segment 3D projection (with the area denoted with red in a(i)) to better highlight the ability of the epoxy dye to completely fill the microfluidic channels and successful pattern a substrate.
3. Conclusions
Here we have demonstrated the process for preserving microfluidic/lab-on-a-chip devices with vibrant colours that can last for at least eleven years. This process enables permanently bonded microfluidic chips to be preserved with a novel epoxy dye, or the epoxy dye to be patterned on substrates with microfluidic chip designs for visualisation and permanent display. By replacing conventionally used food dyes with our developed UV curable epoxy dyes, our process enables microfluidic channels to be filled per usual, but with the colours maintaining their vibrancy for years instead of evaporating after several days.
Furthermore, this process significantly improves the ability to identify details of a microfluidic chip, and encourages the preservation and displaying of microfluidic/lab-on-a-chip research. In addition, most researchers that handle microfluidic chips on a daily basis, will often have chips leftover at the completion of a project and will either discard them or they are left forgotten at the back of a draw. Preserving microfluidic chips would mean that they can serve another purpose, by educating and inspiring future students, collaborators, investors, and spark that inner scientist in us all. Furthermore, the process presented here would enable researchers to showcase their work in display cases or picture frames, as an effective way to communicate and advertise microfluidic art/Art-on-a-Chip, a perfect combination of engineering, science, and art!
4. Experimental
4.1 Materials.
The Sudan dyes were purchased from Sigma-Aldrich: Sudan I (yellow, 103624); Sudan II (orange, 199656); Sudan Blue II (306436); Sudan IV (solvent red, 198102); Sudan Red 7B (fat red, 201618); and Sudan Black B (199664). Solvents used for dissolving the Sudan dyes included isopropanol (BSPPL512, LabServ, Thermo Fisher Scientific), methanol (BSPML868, LabServ, Thermo Fisher Scientific), acetone (BSPAL011, LabServ, Thermo Fisher Scientific), toluene (244511, Sigma-Aldrich), and chloroform (372978, Sigma-Aldrich). UV curable polymers used to produce the epoxy dye included Norland Optical Adhesive (NOA) 72, 81, and 89 (Norland Products). Food dye/colouring was purchased from a local supermarket and used as supplied. Corning® soda lime glass microscope slides were purchased from Sigma-Aldrich. The 3” and 4” prime grade silicon wafers were purchased from WaferPro.
4.2 Epoxy Dye Preparation.
Up to two milligrams of the desired Sudan dye per millilitre of toluene were poured into a glass vial (Arthur Holmes, New Zealand) (Figure 2a). The vial was then manually shaken to ensure that the Sudan dye was thoroughly mixed in the toluene (Figure 2b). Following this, 250 µL of UV curable NOA was
added to the vial and manually shaken until the NOA was thoroughly mixed (Figure 2c). The vials were then left in a fume hood at room temperature until all the toluene evaporated, which took approximately seven days per millilitre of toluene (in a small vial) (Figure 2d). Once prepared, the epoxy dye was kept in a fridge for up to four months, and brought to room temperature prior to use following the NOA manufacturer specifications.
4.3 Photoresist Mold Master.
The photoresist mold masters were fabricated on 4” prime grade silicon using negative tone photoresists (SU-8, MicroChem; ADEXTM and SUEX®, DJ MicroLaminates) following standard fabrication processes previously described.[7, 30, 31] Prior to casting the PDMS microfluidic channels, the mold masters were treated with Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (448931, Sigma-Aldrich) for two hours, to facilitate the removal of the PDMS microchannels.
4.4 PDMS Microchannels.
The PDMS microchannels were produced using standard soft lithography techniques.[30, 32] Briefly, the two-part elastomer PDMS (Sylgard 184, Dow Corning) was prepared at a base to curing agent ratio of 10:1 w/w. The resulting thickness of the PDMS, should be proportional to the duration required for the liquid to completely fill the chip by passive filling.[18, 19] Encapsulating the PDMS, using a ring with an internal diameter of 91.6 mm, resulted in the PDMS having a thickness of approximately two millimetres when using 16.5 g of PDMS. The base and curing agent were mixed together and degassed in a vacuum desiccator (Z119016, Sigma-Aldrich) to remove all the air bubbles. The PDMS was then poured onto the mold master and degassed again to remove any introduced air bubbles. The mold master and PDMS was then placed on a hot plate for two hours at 80 ℃ to cure. After cooling to room temperature, the PDMS was peeled off the mold master and returned to the hot plate for a further two hours at 80 ℃. Once the PDMS cooled to room temperature the inlets were produced, using a one millimetre diameter biopsy punch (ProSciTech).
4.5 Substrate Preparation.
The microscope slides were cleaned with methanol and isopropanol, and then thoroughly dried with nitrogen. When patterning the epoxy dye with intricate structures, such as the leaf nervature and microfluidic text (Supplementary Microfluidic Alphabet), onto a microscopy slide or a silicon wafer, the substrates were dehydrated to promote epoxy adhesion. To dehydrate the substrates they were placed in an oven (>100 ℃) overnight. The substrates were removed from the oven and cooled to room temperature just prior to use.
4.6 Preparing the Microfluidic Chips.
For permanently bonded chips, the microscope slides and PDMS were bonded using oxygen plasma treatment. Briefly, the microscope slides and PDMS were placed in oxygen plasma for 60 s (15 W, pulse ratio: 50, 3 sccm O2; PIE Scientific Tergeo Plasma Cleaner, USA), then removed and brought into contact with each other. The microfluidic chip was placed on a hotplate for two hours at 80 ℃.
To pattern the epoxy dye onto a substrate, the PDMS was temporarily bonded to the glass. To create a temporary bond, the microscope slides and PDMS were cleaned with isopropanol and then deionised water, and thoroughly dried with nitrogen. The PDMS was then placed on the microscope slide and pressed together to create a temporary bond. For more intricate microfluidic chip designs, such as the leaf nervature and microfluidic text, the PDMS underwent silane treatment prior to temporary bonding. Briefly, the PDMS was exposed to oxygen plasma for 60 s to produce a hydrophilic surface (15 W, pulse ratio: 50, 3 sccm O2). The PDMS was then placed in a vacuum desiccator alongside an open glass vial containing a small droplet of trichloro(1H,1H,2H,2H-perfluorooctyl)silane, and placed under vacuum for at least an hour.[12]
4.7 Loading and Curing the Epoxy Dye.
First, the microfluidic chips were placed under vacuum in a desiccator for approximately two hours to enable passive filling of the epoxy dye.[18, 19, 33] After removing the microfluidic chip from the desiccator, a pipette was used to place a small droplet of the desired epoxy dye on the inlets of the microfluidic chip (Figure 2e). Fine tweezers were used to remove any bubbles that occasionally formed in the epoxy dye droplet. This was done to prevent air bubbles forming in the microchannels, which would impact the visual appearance. Following this, the microfluidic chip was left on a flat bench until all the microchannels were entirely filled. Complete filling took anywhere from several minutes to forty minutes. For example, the two millimetre thick ivy nervature microfluidic chip presented in Figure 3c took fifteen minutes to fill with epoxy dye made with NOA 72.
Once the microfluidic chips were filled, the epoxy dye was exposed to UV light at a wavelength of 365 nm (Figure 2f). The curing duration was dependent on the thickness of the PDMS, microfluidic channel geometries, optical adhesive, substrate, and exposure dose of the equipment used. For our microfluidic chips we used a Novacure UV Spot Curing Light Source (EFOS N2001A1) or an OmniCure® S2000 Spot UV Curing System. We placed the microfluidic chips 200 mm below the OmniCure® light source to ensure that all the microfluidic chip was exposed to UV. If this distance was reduced to 100 mm we noticed dye diffusing into the PDMS and if we reduced this distance further we observed considerable fading of
the epoxy dye. When patterning the epoxy onto a substrate (i.e. microscope slide), the PDMS was removed once the epoxy dye was cured to reveal the microfluidic channels on the substrate (Figure 2g). No further curing was required after the microchannels had been removed.
4.8 3D Optical Profiles.
The three-dimensional optical profiles were obtained using a Profilm3D optical Profilometer (Filmetrics Inc., USA), equipped with a 10 × objective (CF Plan 10×/0.30 DI, Nikon, Japan). All the data was acquired using the inbuilt grid scan function in the Profilm3D software 2018 (ver. 3.2.7.2, Filmetrics Inc., USA).
The overall scans had a size of 62 by 36 mm, with a 20% overlap between individual scans, and were obtained by continuous 3D scanning – which took approximately 48 h for each sample. During post processing the scans were stitched together in the Profilm 3D software. The optical profiles were then transferred into Gwyddion (ver. 2.49) for 3-point levelling and image assembly.
Acknowledgements
A. J. M., J. T. N., and L. P. L acknowledge the Intel Scholars Program. We acknowledge Dan Malleo for imaging the microfluidic Golden Gate Bridge and integrated microfluidic cell culture and lysis on a chip.
R. S. thanks the New Zealand National Science Challenge - Science for Technological Innovation for a Post-Doctoral Fellowship. S.O. thanks the MacDiarmid Institute for a Ph.D. Scholarship. R. S, S. O., and V. N. thank Helen Devereux and Gary Turner for technical assistance in the University of Canterbury Nanofabrication Laboratory.
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