Room temperature roll-to-roll additive manufacturing of polydimethylsiloxane-based centrifugal microfluidic device for on-site isolation of ribonucleic acid from whole blood
Trung Hoang a c, Han Truong a c e, Jiyeon Han a c e, Saebom Lee b, Jihyeong Lee b, Sajjan Parajuli d e, Jinkee Lee a b, GyouJin Cho
Polymer-based lab-on-a-disc (LoaD) devices for isolating ribonucleic acid (RNA) from whole blood samples have gained considerable attention for accurate biomedical analysis and point-of-care diagnostics. However, the mass production of these devices remains challenging in manufacturing cost and sustainability, primarily due to the utilization of a laser cutter or router computer numerical control (CNC) machine for engraving and cutting plastics in the conventional prototyping process. Herein, we reported the first energy-efficient room-temperature printing-imprinting integrated roll-to-roll manufacturing platform for mass production of a polydimethylsiloxane (PDMS)-based LoaD to on-site isolate ribonucleic acid (RNA) from undiluted blood samples. We significantly reduced energy consumption and eliminated thermal expansion variations between the mold, substrate, and resists by accelerating the PDMS curing time to less than 10 min at room temperature without using heat or ultraviolet radiation. The additive manufacturing technology was applied to fabricate a multi-depth flexible polymer mold that integrated macro (2 mm) and micro-sized (500 μm) features, which overcomes the economic and environmental challenges of conventional molding techniques. Our integrated R2R platform was enabled to print adhesion-promoting films at the first printing unit and continuously in-line imprint with a high replication accuracy (99%) for high-volume manufacturing of a new centrifugal microfluidic chip with an enhancement of mixing performance by integrating an efficient mixing chamber and serpentine micromixer. This research paved the way for scalable green manufacturing of large-volume polymer-based microfluidic devices, often required in real-world sample-driven analytical systems for clinical bioanalysis.
Room-temperature PDMSCentrifugal microfluidicRNA extractionRoll-to-roll nanoimprint lithographySustainable manufacturing
THE FABRICATION OF LoaD DEVICES
A typical LoaD consists of a multilayer platform made from thermoplastics, such as polycarbonate (PC), poly (methyl methacrylate) (PMMA), polystyrene (PS), cycloolefin polymer (COP), assembled by adhesive or through hot embossing and injection molding methods [9–11], which is cost-effective for high-volume manufacturing process. These devices can also be fabricated through a laser or router computer numerical control (CNC) machine for engraving and cutting plastics, in conjunction with the utilization of plotter machines to cut the adhesive film [12–14]. However, for CNC-manufactured microfluidic devices, the surface becomes extremely rough, leading to slow and inaccurate fluid flow and bonding inhibition as well as non-specific binding which can negatively impact the performance of microfluidic devices. Also, the utilization of these subtractive manufacturing systems has been constrained by the capability of the cutting tools, making the fabrication process of micro-scale features even more expensive, lengthy, and complex, limiting the fabrication throughput, and presenting challenges for mass production ability. Among polymer-based materials, polydimethylsiloxane (PDMS) has been widely used to fabricate microfluidic devices via soft lithography [15–19]. The use of PDMS in manufacturing LoaD devices has been considered as an alternative strategy to the CNC-based one due to its capability to reduce production costs, increase flexibility, facilitate ease of fabrication, and permit rapid prototyping without the use of harmful etching chemicals.
ROLL-TO-ROLL LITHOGRAPHY AND OVERCOMING ITS CHALLENGES
Roll-to-roll (R2R) nanoimprint lithography is considered as an up- and-coming alternative to traditional manufacturing methodologies, owing to its ability to achieve high-throughput production and thus facilitate its application at an industrial scale . PDMS was first demonstrated to be R2R processable by Ahn and Guo  (2008) for sub-micrometre test structures on a polyethylene terephthalate (PET) substrate. Later, Hiltunen proposed R2R fabrication of integrated PDMS-paper microfluidics for molecular diagnostics . However, both techniques relied on thermal imprinting to cure PDMS resist by heating the imprinting roll to a high temperature with a large amount of energy. This requires roll modifications with complex and expensive auxiliary systems such as piping networks to circulate the heating liquids, the pre-heating and heating units, cooling system as well as temperature monitoring and control system, which increase waste heat and energy consumption, consequently causing greenhouse gas emissions. Additionally, the mismatch in thermal expansion coefficients between the mold and substrate results in the generation of lateral strain, which degrades the quality of the imprinted patterns and the lifetime of the mold . Also, the molds used in these proposed methods were limited to a micrometre scale, making them impossible to apply for the fabrication of large-volume LoaD which requires millimetre-scale thickness. To overcome those issues mentioned above, we developed the first room-temperature printing - imprinting integrated roll-to-roll (R2R) in- line manufacturing platform for mass production of a PDMS-based LoaD for on-site RNA isolation from whole blood samples with low cost, less energy consumption, and less by-products. In this work, multi-depth master stamps were first fabricated by using a 3D printer, overcoming the challenges of conventional CNC-based and photolithographic molding technology. The large area flexible polymer shim was fabricated by using a rubber sheet to stick PDMS molds which were replicated from 3D-printed stamps. For mass producing the large volume LoaD by R2R technology, the printing unit was integrated in-line with an imprinting platform for coating an adhesion promoter onto PET substrate to facilitate the demolding process. We incorporated well-defined compounds into commercial PDMS Sylgard 184 formulations to accelerate the curing time of PDMS at room temperature enabling the success of a low-temperature R2R imprinting process which helped to reduce heat waste and energy consumption. Finally, as a proof-of-concept study, novel LoaD devices with a high enhancement of mixing performance were sustainably manufactured by our green R2R platform. For the first time, the utility of these R2R-manufactured LoaD devices was demonstrated by isolating RNA from undiluted blood samples.
2.1Fabrication of negative 3D-printed mold
Our LoaD device comprises two layers: the designed patterns layer and a blank PDMS as a coverlid. The 3D model of the top part was designed by using SOLIDWORKS software, which was then exported as an STL file. We used the Utility Version 6.3.0t3 software to convert STL files into sliced files with appropriate layer heights. Printing options were optimized for high-resolution printing of mold features by using a 3D printer (PR110-385 from CADworks3D company) utilizing a 385 nm light source with a printing area of 110 × 62 × 120 mm and XY resolution of 40 μm. In this work, we printed all masters using the 3D printing protocol with the following parameters: a UV projection time per layer: 9.5 s and a z-axis resolution: 50 μm. It has been shown that 3D-printed templates interfere with the curing of PDMS due to uncured resin remaining on their surface . Therefore, a post-print surface treatment technique is commonly necessary to overcome the aforementioned issues and prevent the adhesion of PDMS to the 3D-printed mold. A treatment method involving coating the template with a protective ink using airbrushing was reported by Comina et al. . The group claimed this technique requires much effort for achieving optimal results. Ho et al. proposed another complicated surface treatment method for 3D printed templates, including heating, plasma treatment, and surface silanization . However, cracks were formed in the template during the heating process. To avoid these time-consuming, nonreplicable, and ineffective surface treatment methods, we fabricated master molds directly via the DLP 3D printing method utilizing a novel resin formulated by Creative CADworks company (CCW Master Mold for PDMS devices), composed of methacrylated oligomers and monomers. After removing printed molds from the picker, they were subjected to a thorough rinsing procedure utilizing isopropanol. Subsequently, an air nozzle was employed to eliminate residual resin from edges and within extremely fine features. Finally, we postcured the molds by exposing each part to UV light at a wavelength of 405 ± 5 nm within a curing chamber, and then, the resulting molds were employed in our imprinting works.
2.2 Fabrication of positive PDMS mold
To fabricate positive PDMS mold, we prepared a mixture including PDMS base and curing agent in the ratio (W/W) of 10:1 (Sylgard 184 from Dow Corning, MI, USA). The mixture was mixed and defoamed with a centrifugal mixer (Thinky Mixer ARE-310) for 3 and 2 min, respectively, and then poured onto the 3D-printed molds without undergoing any surface treatment process. Later, we cured it in the oven for 4 h at 60 °C. Subsequently, the cured PDMS was carefully detached from the molds and cleaned thoroughly with isopropyl alcohol (IPA) and ethanol at least three times, followed by air drying between each wash. To be used as the mold for the R2R imprinting process, we applied a conformal coating of parylene C which serves as an anti-adhesion layer for demoulding these positive PDMS replicas . Finally, a flexo plate with 5 mm thickness was cut with precise squares, used as the substrate for inserting PDMS positive molds, and wrapped to form a sleeve for preparing the imprinting cylinder.
2.3. PDMS formulations for R2R imprinting process
We formulated a new recipe for fast curable PDMS at room temperature (named Room-Temp PDMS in this paper) by adding compounds (the Ashby–Karstedt catalyst and tetrakis (dimethylsiloxy) silane) into commercial PDMS Sylgard 184 formulations . SYLGARD™ 184 Silicone Elastomer and curing agent were supplied by Dow. Ashby–Karstedt catalyst (platinumcyclovinylmethyl-siloxane complex; 2 % Pt (0) in cyclomethylvinylsiloxanes) and Tetrakis (dimethylsiloxy) silane (TDS) were supplied by Gelest. We used a ratio of 10:1 (w/w) for the elastomer and curing agent to make the standard Sylgard 184 framework. In this study, these compounds were always added following the optimized recipe in exact order: (1) TDS 2 wt%; (2) Elastomer base 88.95 wt%; (3) Curing Agent 8.9 wt%; and (4) Ashby–Karstedt catalyst 0.15 wt%. After all the chemicals had been added, we placed the mixture in Reactor-Ready included circulator Huber Ministat 230, both were supplied by Radleys company (Fig. S8) for continuous mixing at 500 rpm and cooling at −15 °C to prolong the lifetime of PDMS for the imprinting process. After mixing for 1 h, we turned off the mixer and set the reactor at a vacuum of 0.2 psi for degassing in 1 h meanwhile maintaining cooling during the whole process. For comparison, we also prepared the standard PDMS Sylgard 184 as control samples (named Sylgard 184 in this paper) and the reference samples (named Vinyl-terminated PDMS in this paper) by optimizing the recipe of a fast, thermal-curable liquid resist  based on a modified PDMS. The formulation for this reference sample consists of three components: vinyl-terminated PDMS was supplied by Gelest, poly (dimethylsiloxane-co-methylhydrosiloxane) trimethylsilyl terminated which acts as a crosslinker and platinum (0)-1,3- divinyl-1,1,3,3-tetramethyldisiloxane complex solution as a catalyst, both were supplied by Sigma-Aldrich. In our optimized recipe, we mixed a ratio of 5:1 (w/w) for the vinyl-terminated PDMS and crosslinker, then added Pt catalyst into the mixer at a concentration of 470 ppm. This formulation effectively accelerated the curing time of this PDMS-based resist at room temperature, making it possible to be used for the R2R imprinting process.
2.4. Roll-to-roll imprinting process and chip assembly
The replication by the R2R imprinting process was conducted at room temperature through an imprinting unit as illustrated in Fig. S1. First, we treated a roll of 150 mm wide PET substrate with an adhesion promoter (Wacker Primer G790) by gravure printing method with a speed of 3 mm/s for five layers then dried in line at room temperature. The coated PET substrate remained as transparent as the original one, as shown in Fig. S2a. The premixed PDMS was deposited to the gap between the imprinting roll and pressure roll during the R2R imprinting process. We operated the process at room temperature with optimized pressure by testing 1 to 10 kgf/cm2 under an imprinting speed of 0.3 mm/s. After coming out from the imprinting unit, imprinted chips on the PET substrate were collected at the rewind roller and were applied to a cutting process for making single devices. The coverlids were simply fabricated by pouring our room-temperature PDMS into a 3D-printed square mold with a dimension of 55 × 55 mm, resulting in unstructured PDMS layers with a uniform thickness of 1 mm. The inlets and outlets for each chamber were manually punched. After that, a 3 mm diameter circular disc magnet which was supplied by First4Magnets, was preloaded into the mixing chamber of each device, and lids were then bonded onto the imprinted layer with a plasma treatment method.
2.5. Replication accuracy and material characterization
Replication accuracy. Dimensional analysis of the 3D printed mold, PDMS mold, and the R2R imprinted LoaD was performed with an industrial microscope Olympus BX53 M.
Optical properties measurement. Autofluorescence measurements of three types of PDMS and glass were conducted by using a Nikon Eclipse Ti2 microscope with 4 different excitation wavelengths. The optical transmittance was measured on all three PDMS types: Sylgard 184, Modified Vinyl-terminated PDMS, and Modified Sylgard 184 using a complete setup of a 508 PV™ UV–visible–NIR Spectrophotometer integrated with a bright field microscope Olympus BX53 M. For both measurements, we prepared 3 samples for each PDMS by pouring uncured PDMS into the circle Petri dish with a diameter of 55 mm with a uniform thickness of 2 mm and curing at 80 °C. All the samples were cleaned by sonicating in ethanol for 30 min and then dried with a nitrogen gun before analysis. To check the discoloration phenomenon of these materials, we prepared 5 samples for each type of PDMS in the same way as mentioned above, but all were cured at room temperature. After fully curing all samples, we aged them thermally in 5 isothermal ovens respectively at 20 °C, 40 °C, 60 °C, 80 °C, and 100 °C for 1 h before analyzing.
Contact angle measurements. We measured the static contact angle for each type of PDMS by using a Drop Shape Analyzer (DSA100) from Krüss. Measurements were conducted using an automated drop dispenser and deposition device to dispense a 2 μl drop of deionized (DI) water on the material surface. The static contact angle was calculated using computer software.
Mechanical characterization techniques. Tensile strength tests were conducted using the Instron Electropuls E3000 testing system. All samples for three types of PDMS were prepared following ASTM D-412 standards which were cut into dumbbell shapes, referred to as dogbone, using a type-D specimen die. The samples were put into the uniaxial grips and dragged at a speed of 3.84 mm/s until they reached a breakpoint . We performed the tests on five trials for each sample then the engineering stress and strain were reported as averages and corresponding standard deviations.
2.6. Nucleic acid design and reagents
Complete genomes of SARS-CoV-2 Wuhan wildtypes (accession MN908947.3) were retrieved from NCBI (https://www.ncbi.-nlm.nih.gov/). Forward Primer -ACAGGTACGTTAATAGTTAATAGCGT and Reverse Primer -ATATTGCAGCAGTACGCACACA were purchased from Cosmogenetech Inc., (Seoul, Korea). The experiment used SARS-CoV-2 mRNA spiked in human whole blood (Innovative Research,USA) for further analysis. RNA purification reagents were purchased from MagListo 5 M viral DNA/RNA extraction (Bioneer). The buffer solutions (proteinase K, virus binding buffer (VB)), washing buffer (VWM1, RWA2, and WE buffer), elution buffer (ER buffer), magnetic nanobead (MNPs) were preloaded into the chip with the volume: 20 μl proteinase K, 200 μl VB, 400 μl absolute ethanol, 50 μl magnetic nanobead, 500 μl washing buffer, 100 μl ER buffer. 200 μl of blood was added at the beginning of the operation. Polymerase chain reaction (PCR) was performed using the following protocol: reverse transcription (42 °C for 5 min, 95 °C for 10 s), PCR test (40 cycles of 95 °C for 5 s, 60 °C for 30 s), melting curve analysis (95 °C for 0 s, 65 °C for 15 s, 95 °C for 0 s) with 20 μl of final PCR volume (One Step TB Green® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (Takara Bio, Tokyo, Japan).
In our LoaD devices, we utilized ferro-wax valves which were made by mixing paraffin wax (327,204, Sigma-Aldrich, Inc., St. Louis, MO, USA) with Ferrofluid (EFH1, 60 cc, Ferrotec, Santa Clara, CA, USA) in the ratio of 1:1 b y mass and stirring the mixture at 65 °C on a hotplate for 12 h.
We developed the first sustainable R2R additive manufacturing platform for fabricating PDMS-based centrifugal microfluidic devices at room temperature without using heat and light sources that minimize the emission of greenhouse gas and hazardous by-products (Fig. 1). In this work, we used the continuous in-line printing and imprinting units of our R2R system, illustrated in Fig. 1a. By utilizing additive manufacturing, we introduced sustainable fabrication techniques for a deep-depth flexible shim that addressed the issues of traditional molding technologies (Fig. 1b). As illustrated in Fig. 1c, LoaD devices were successfully manufactured at ambient condition by using the new fast-room temperature-curing PDMS based on Sylgard 184 to eliminate the need for heat and light sources which are often used in conventional R2R imprinting process. The process of on-chip isolation of RNA from whole blood using our R2R imprinted LoaD was illustrated in Fig. 1d. Before the imprinting process, the PET substrate was uniformly treated by R2R gravure printing unit with an adhesion promoter that effectively assisted the demoulding of structures with a large thickness (∼2 mm) (Fig. 1e and f). Finally, we successfully demonstrated the high throughput and sustainable manufacturing platform for producing the LoaD devices (Fig. 1g).
Fig. 1. Overview of the room-temperature roll-to-roll imprinted PDMS-based centrifugal microfluidic devices. (a) Roll-to-roll additive manufacturing platform; (b) Enlarged image of R2R imprint unit; (c) Rewinder unit for collecting imprinted LoaD devices; (d) Illustration of on-chip isolation of RNA from whole blood using our R2R imprinted LoaD device; (e) and (f) R2R gravure coating unit; (g) Mass production of PDMS-based centrifugal microfluidic devices on PET substrate.