1. Introduction
Microfluidics refers to the science that allows the study and manipulation of fluids on the micrometer scale [
1]. Over the last two decades, microfluidic systems have been established as promising platforms for many lab-on-chip (LOC) applications in several research fields, such as chemistry, biology, medicine, and engineering. The main advantages of the microfluidic approach lie in the ability to control liquids under a laminar regime, reduction in the amount of reagents and sample volume, shorter analysis time, reduction in cost, and portability [
2]. Polydimethylsiloxane (PDMS) is the most used material for the fabrication of microfluidic devices as it is optically transparent, biocompatible, easy to fabricate and its manufacture does not require high capital investment or cleanroom conditions [
3]. One key step for PDMS chip manufacturing is to bond the PDMS replica with another substrate (usually glass) to assemble the microfluidic channels and possess high bond strength and stability. An extensive and critical review published by [
4] gathers many methodologies for irreversible or reversible PDMS sealing to PDMS or other different planar structures—such as silicon, glass, or other polymers—and different ways to make fluidic or electrical connections. Among all sealing methods described to date, oxidation using oxygen plasma treatment is the most common method [
5]. Although irreversible sealing methodologies are useful due to their strong bonding and ability to support high pressures, they present some disadvantages, such as low reusability and difficult internal accessibility into microchannels.
On the other hand, the reversible sealing of microdevices allows easy disassembling, cleaning, reassembling, and reduction of manufacturing time. PDMS chips can directly attach to other PDMS or glass slides through Vander Waals forces. For example, Park et.al. [
6] fabricated microfluidic culture platforms using reversible bonding by lightly touching the PDMS on a glass coverslip previously coated with poly-L-lysine [
6]. However, this procedure is only suitable for low pressures (less than 35 kPa), and chips are prone to debonding or leaking. To overcome this drawback, other techniques with the ability to support higher pressures (up to 100 kPa) have been proposed. For example, sealing by vacuum suction [
7,
8,
9] consists of crossing a channel air network around the main microchannels where the vacuum is applied by aspiration. Nevertheless, this application requires additional working space for the vacuum source, as well as an additional microchannel network around the main microdevice. A magnetic seal is another technique proposed for reversible bonding by applying an external and controlled magnetic force [
10,
11]. More recently, Tsao and Lee [
12] fabricated an iron oxide magnetic microparticle PDMS composite material, offering the possibility to cast in an opaque-view or a clear-view. However, with the opaque-view material, optical detection was not possible, and the surface roughness increased with respect to native PDMS [
13]. In general, reversible sealing by magnetic forces allows a uniform and long-range of pressures depending on the applied magnetic field strength. Nevertheless, the presence of slab-shaped magnetic material restricts the device microchannel network to very simple geometries [
12]. Commercial adhesive tapes for the reversible bonding of microfluidic chips have also been reported, where the tape is placed under a PDMS chip and baked at 65 °C for 2 h. The tape can be peeled off, and the microdevice can be reused after it has been washed; in contrast, adhesives increase the possibility of contamination [
12].
In the current paper, we present a novel modular microfluidic platform that is easy to assemble and allows the reversible bonding and reuse of the microfluidic device. This innovative and mechanical approach for reversible sealing in a microfluidic device consists of placing the polydimethylsiloxane (PDMS) chip between two planar poly(methyl methacrylate) (PMMA) plates and applying mechanical and uniform pressure. The reversible seal proposed in this work is mechanical clamping, a technique that has been explored in general for glass–PDMS–glass sandwich [
14]. In addition, a molding method is shown to manufacture microfluidic devices by casting PDMS in a reproducible and safe way (REPSAF). This method also uses less PDMS and produces polished walls.
As a proof of concept, we validated the method by applying PDMS chips for droplet generation. Droplets are produced by breaking the surface tension between a continuous phase (oil) and a dispersed phase (water) within multiple models of devices that allow controlling flow mixtures and droplet size [
15,
16]. However, a set-up in many of the applications requires many devices, which implies great expenditure in materials and manufacturing time. We address this issue with the chip on/off configuration to enable reusability obtained with the new methodology.
2. Materials and Methods
2.1. Mechanical Pressure Set-Up
The mechanical pressure assembly consists of two PMMA plates (PLEXIGLAS
®).-The supplier uses a manufacturing protocol combining bulk emulsion and solution emulsion). The plates were made to measure corresponding to a base and a lid, with dimensions of 5 × 85 × 70 mm.
Figure 1 shows how the microchannels containing PDMS and the glass base of the chip are placed between the PMMA plates. Four JM
® brand stainless steel screws of same size were used to generate the same pressure (D for certain experiments: 8.16 mm and L: 17 mm) The screws were fixed to reversibly join the microdevice and apply mechanical pressure. The screws are located at 50 mm in width and 55 mm in length from each other. The PMMA top plate has one inlet hole and one outlet hole into which 24 mm long and 3 mm wide polytetrafluoroethylene (PTFE) hollow screws are inserted and then connected to polyvinyl chloride (PVC) tubing 18 mm long and 3 mm wide. These tubes are connected to a syringe pump (ADOX 22a)
2.2. Design and Fabrication of Microfluidic Device
The droplet-forming microdevice (
Figure 2) was photolithographed in a high relief mold with the desired pattern on a 700 µm thick silicon wafer (Virginia Semiconductor, Inc., Fredericksburg, VA, USA), using the negative resin SU-8 (MicroChem, Round Rock, TX, USA). The microchannels have a final height of 150 µm. Next, the mold was placed under vacuum with trichloro (1H, 1H, 2H, 2H-perfluoro-octyl) silane (Sigma, St. Louis, MO, USA) for 1 h to protect the SU-8 resin from detachment by releasing PDMS from the mold. The PDMS was mixed with the curing agent in a 10:1 ratio and the mixture was placed under vacuum for 1 h to remove air bubbles. Next, the mixture was poured back under vacuum for 1 h and cured in an oven at 70 °C for 70 min. The PDMS was molded, and the fluidic connection ports were constructed by drilling holes in the PDMS with a hole punch (21-gauge, internal diameter of 0.51 mm). Finally, the PDMS device was assembled with a glass base.
2.3. Deformation and Flow-Rate Measurement
Testing and validation of the newly manufactured microdevice has been conducted for changes in the structure (channels) and the flowrates at various pressures. Channel deformation produced by compression has been measured with a stereomicroscope (BioTraza, Zhejiang, China) 2,3 × magnification. The stereomicroscope has been coupled to a Canon EOS 600D camera HD (1080p 1920 × 1080) recording at 29.97, 25, or 23.976 frames/s. The deformation is determined by taking serial images at different pressures focused on a single design structure. Pressure on the microfluidic device was measured with a force-sensing resistor (Interlink Electronics FSRTM 400—Interlink Electronics, Lake Forest, IL, USA) coupled to a multimeter UNI-T (UT39A) located at the right corner below the chip. ImageJ software [
17] was used for measuring structure length (pixels) changes at different pressures.
Flow rates at different pressures were measured using aqueous suspensions of Acid Blue 1 dye 0.648 mg mL−1 (Sigma-Aldrich, St. Louis, MO, USA). Video images were analyzed to measure the time employed by the front of the dye to travel a fixed microchannel distance.
2.4. Droplet Generation
A flow-centered emulsion droplet microfluidic device was designed, constructed, and used for the generation of monodisperse micrometric-sized droplets, consisting of two inlets and one outlet for droplet recovering. The internal phase, 2% blue aniline solution (% w/v) was pumped (AcTIVA Infusion ADOX A22, CABA, Argentina) at a constant rate of 0.80 µL/min, the continuous phase mineral oil (Sigma-Aldrich, St. Louis, MO, USA) with SPAN 80 surfactant (Sigma-Aldrich) (5% w/v) was pumped at a rate of 1.15 µL/min.
2.5. Device Reuse
To achieve optimal reuse of the device, a method based on continuous washing is proposed. In an airtight container, the device was immersed in a solution containing a commercial degreaser (CLEAN LAB®) for 10 min, and then this device was rinsed with distilled water. The next step included immediate immersion of the device in a new commercial degreaser solution for another 10 min, followed by a new washing with distilled water. Later, the device was immersed in absolute ethanol (96%) for 10 min in a new airtight container, followed by a distilled water rinse. The device was immersed again in 70% ethanol for another 10 min. Finally, this device was rinsed with Milly Q quality water and subjected to heat (70 °C) for 2 h using Thermo Electron Precision oven.
2.6. Droplet Images
The Olympus BX40 microscope with 10X lenses and a Canon EOS 600D digital camera attached to the microscope was brought out to view and acquire video of drop formation. Images were obtained from a stack of multiple microscope acquisitions on the surface of the device. To analyze the size distribution of the produced droplets, the area occupied by 100 drops in each experiment was subsequently measured using image J processing software with the specific plugin to visualize circular objects [
18]. Additionally, the average droplets diameter and standard deviation was reported.
4. Conclusions
In this work, a reversible and low-cost microfluidic sealing methodology is proposed. It consists of mechanically holding the chip between two PMMA plates. This proposed methodology does not require clean room facilities for device cleaning, nor does it require plasma bonding, and it is easy to perform. Furthermore, the proposed manufacturing method allows simple unmolding, as well as uniform dimensions throughout the entire PDMS chip.
In addition, it is possible to save a considerable amount of PDMS, since the methodology limits waste due to its closed edges for each model. The methodology increases the versatility of the use of microfluidic devices since it does not imply an irreversible union.
In our work, we verified the reliability and performance of the droplet-forming microdevice that has multiple uses, such as the recovery of droplets with chemical compounds, drugs, the recovery of specific clone cells, and hydrogels for long-term culture.
It is also important to note that the REPSAF system not only applies to droplet-forming microdevices, but it can also be used with cell culture chips, diluters, and applications such as EOR assisted oil recovery. In addition, the system can be used with different materials such as glass, plastic, or PMMA for several applications.