Rapid and cost-effective prototyping of microdevices for biomedical applications
Creators: name: Ye, Mengguang
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: micro-electromechanical systems
microfluidic devices
3D printing
cost-effective manufacturing
AST technology
biomedical applications

description: Microfluidics enables the manipulation of small fluid volumes and facilitates the study of cellular processes with high resolution. These systems can significantly reduce the reagents and time required for various laboratory-based tests. The concept of using microfluidic devices for precise fluid manipulation at the micro- or nanoscale originated in the semiconductor industry. It was later recognized as a valuable tool and further developed in the microelectromechanical systems (MEMS) field. Today, microfluidic devices are extensively utilized across various domains, including cell culture, drug delivery, chemical and biological synthesis, flow cytometry, and environmental monitoring. In this thesis, the focus will be on the application of microfluidics in biomedical research. Initially, microfluidic devices were manufactured using techniques inherited from the electronics industry, such as anodic bonding, etching, thin film deposition, and photolithography. Since then, a variety of methods have been developed to offer rapid and robust fabrication of microfluidics, including soft lithography, injection molding, and 3D printing. Replica molding, also known as soft lithography, was a commonly utilized method for the fabrication of microfluidic devices. This method involves the fabrication of a replication master, casting, and bonding. Although soft lithography exhibited advantages in the fabrication of microfluidic devices, mold fabrication required photolithography, which is costly, time-consuming, and necessitated designated facilities, such as a clean room. Additionally, poly(dimethylsiloxane) (PDMS), a representative material in microfluidic device fabrication due to its affordability, transparency, biocompatibility, low autofluorescence, and ease of production, tended to deform under high fluid pressure, leading to less reliable performance. As an alternative, 3D printing allows for rapid prototyping and customization of complex microfluidic structures and have the potential to significantly reduce the cost and time required for device fabrication, as well as the need for designated facilities. However, 3D printed devices are typically opaque, and the commonly used resins were cytotoxic, rendering the devices unsuitable for biomedical applications. In chapter 2, the 3D printing approach was optimized to produce transparent, biocompatible microfluidic devices. The issues of cytotoxicity, opacity, and low printing fidelity, which limited the use of 3D printing in biomedical applications, were eliminated. After optimization, the 3D printing approach allowed for the direct and rapid manufacturing of complex microfluidic structures without using molds for PDMS casting. One key advantage of our 3D printed microfluidic devices was their transparency, permitting direct use of the device for optical assessments. Additionally, an intensive washing protocol was developed to render the 3D printed microfluidic devices highly biocompatible, making them particularly useful for biomedical applications. Eventually, a range of microfluidic devices with various biomedical applications was tested, indicating they are a promising alternative for the production of microfluidic devices in both research and industrial settings. Overall, after optimization, the 3D printing approach demonstrated a rapid and cost-effective method for manufacturing microfluidic devices for biomedical applications. In chapter 3, the 3D printing approach developed in the first chapter was applied to fabricate an inertial spiral microfluidic device for highly efficient and high-throughput bacterial separation from whole human blood. Direct detection and separation of bacteria in bloodstream infections (BSIs) have the potential to significantly reduce the turnaround time for sepsis diagnosis, providing information on antibiotic susceptibility. This optimization could enhance patient care and guide therapy. The functionality of the 3D-printed spiral microfluidic device was validated, demonstrating high throughput and high separation efficiency under conditions of clinically relevant low bacterial concentrations, which are critical performance parameters from a clinical application perspective. This 3D printed microfluidic device demonstrated an average separation efficiency of 82% for Escherichia coli in whole blood samples. The separation functions effectively even at a bacterial concentration as low as 10 CFU/ml. Of note, our device can be manufactured rapidly and cost-effectively using 3D printing technology in under 30 minutes and can process 1 ml of whole human blood within 11-17 minutes, depending on the blood dilution level. The fast-processing time and the reliability to separate bacteria from a significant volume of patient blood enable the use of this technique in downstream analyses of potential genomic information and antibiotic susceptibility patterns. In chapter 4, a novel 3D-printed droplet microfluidic device featuring multi-layer structures was introduced to facilitate antibiotic susceptibility testing (AST). The global threat of antibiotic resistance presented a significant challenge and was associated with an increasing risk of death. There is an urgent need for rapid AST technology to mitigate mortality risks by avoiding delays in essential treatment. The droplet microfluidics offered rapid AST by utilizing significantly smaller sample volumes than traditional methods. Single bacterium cell was encapsulated in a small aqueous droplet, where this miniaturized reaction volume elevated the local concentration of bacteria and facilitated a more rapid determination of antibiotic effectiveness, thereby advancing the efficiency of AST processes. The design of our droplet microfluidic device featured four layers in height regions and a 25 μm flow-focusing nozzle. These multi-height regions optimized flow resistance, enabling stable and rapid droplet generation. Unlike the weeks-long process required by conventional soft lithography to obtain microfluidics with multi-layers, 3D printing can fabricate a microfluidic device with multilayer structures within 1 hour, offering high resolution and fidelity even when compared to those produced by conventional soft lithography. Overall, this device was designed for the encapsulation of single bacterium within an 18 pL droplet, utilizing a rapid resazurin-based fluorescence growth assay to detect fluorescent signal intensity after 1.5 hours incubation. To demonstrate the effectiveness of the 3D-printed droplet microfluidic device in performing AST, the device was employed to evaluate the antibacterial effects of ampicillin and ciprofloxacin on E. coli growth. The E. coli strain was determined to be resistant to ampicillin and susceptible to ciprofloxacin. In Chapter 5, the 3D-printed droplet microfluidic device established in Chapter 4 was further explored to generate three different types of hydrogel beads: PEGDA hydrogel beads, PEGDAPEGSH hydrogel beads, and core-shell hydrogel beads. Confining a single cell within a small aqueous droplet can rapidly accumulate cell secretions or biomass to a detectable level. However, aqueous droplets are not ideal for long-term cell culturing, as the small volume can lead to rapid nutrient depletion and waste accumulation, adversely affecting cell behaviour. To address the issues arising from the lack of solution exchange between the interior environment of the droplet and the external environment, hydrogels as inherently porous materials for encapsulating cells and enclosing biomolecules have been studied. Additionally, the passive filtration system at the inlets was redesigned to feature five arrays of pillars with 50 μm gaps. This modification effectively prevents blockages at the flow-focusing nozzle and ensures reliable operation. This improvement addresses the shortcomings of the previous design, which had larger 150 μm gaps that allowed dust and fibres to cause blockages at the flow-focusing nozzle. Overall, this thesis provides a comprehensive investigation into the use of 3D printed microfluidic devices in biomedical applications, highlighting their benefits in the fabrication process, including rapid prototyping and the customization of complex microfluidic structures. Compared to other 3D-printed microfluidic devices in the literature, the microfluidic devices presented in this thesis offer high optical transparency and resolution, even when compared to those produced by conventional soft lithography. Given the low cost and short turnaround time associated with fabricating microfluidic devices using 3D printing, this thesis may inspire future research utilizing microfluidic devices and broaden the application of microfluidics across various fields.