LAURA GALLOWAY, MARKETING DIRECTOR, BOSTON MICRO FABRICATION (BMF)
Microfluidics, the study of fluid behaviour in microscale channels, is becoming increasingly important for medical and laboratory diagnostics. For handling fluid samples of one part per million (1 ppm) or less, microfluidic devices (MFDs) provide a quick, safe and cost-effective way to sequence DNA (genomics) and RNA (transcriptomics). These tiny diagnostic devices are also used to characterise proteins (proteomics) and metabolites (metabolomics). With lab-on-a-chip (LOC) technology, extremely small fluid volumes can be measured in femtolitres (fL), a quadrillionth of a litre.
Even before the COVID-19 pandemic, MFDs were used in many biological, pharmaceutical and medical applications, and for environmental analysis and food and agricultural research. Today, there are several microfluidics areas where 3D microprinting, a form of additive manufacturing (AM) that produces microscale objects, offers promising applications for MFD prototyping and production. By contrast, traditional fabrication and assembly technologies such as injection micromoulding, soft lithography and lamination present limitations in terms of cost, turnaround times and design freedom.
Traditional MFD fabrication methods versus 3D microprinting
Traditional MFD fabrication methods include injection micromoulding, soft lithography and lamination, although these have significant disadvantages. Injection micromoulding requires precision tooling, which can be expensive to machine and take weeks or even months to arrive; soft lithography limits researchers’ ability to produce complex 3D channels, which is a concern for MFD designers who want to create microchannels with diameters less than 100 µm and high aspect ratios; and lamination requires cutting the desired microfluidic features into layers and then bonding these individual layers together to form a functioning unit in a multi-step process that is both labour-intensive and time-consuming.
A microfluidic (MFD) chip produced on a microArch printer.
3D microprinting can produce intricate objects like MFDs, but few technologies can create microscale components with fine features and tight tolerances at the required resolution, desired speed and with polymers that have the required properties. Projection microstereolithography (PμSL), a patented stereolithography (SLA) technology that has been commercialised by Boston Micro Fabrication (BMF) in the form of its microArch range of 3D printers, strikes the right balance between speed and precision. PμSL supports the use of polymers with application-specific properties, such as high temperature resistance, chemical resistance and biocompatibility, and can produce complete MFDs with a good quality surface finish for proper fluid flow. Moreover, these tiny devices can be used for either prototyping or end-use. In addition to fabricating complete MFDs, PμSL can support the production of high-precision microtooling for soft lithography, capable of fabricating small components down to 2 µm resolution and +/-10 µm accuracy at scale.
An MFD chip produced on a microArch printer.
Single-cell analysis
Single-cell analysis rapidly assays single cells using droplet microfluidics, the generation and manipulation of discrete droplets through immiscible, multiphase flows inside microchannels. Applications include the isolation of cancer cells for downstream testing and drug testing, and the discovery of new cell subtypes and biomarkers. To support cellular analysis, individual cells are encapsulated in droplets with a lysis buffer and other reagents. When a cell explodes, its internal constituents are exposed for conjugation with these other reagents.
The MFDs for single-cell analysis contain tiny channels that can be fabricated using injection micromoulding, photolithography and etching, precision machining or hot embossing. MFD components can be assembled into complete devices using adhesive bonding, heat staking, solvent bonding, lamination or laser welding. PµSL can replace traditional fabrication and assembly methods because it meets important technical requirements for single-cell assays. Furthermore, PµSL is significantly faster than traditional fabrication and assembly methods and can reduce both costs and risks.
The technical requirements for single-cell analysis include the use of hydrophilic microchannels that ensure minimal to no diffusion between droplets and that have water contact angles of <30 and >150°. Since these tiny channels are exposed to water-based and oil-based reagents, the channel material must provide requisite chemical compatibility. In vitro cytotoxicity is also important because cells pass briefly through the channels. In addition, the channel material requires sufficient optical transparency to support droplet analysis during characterisation and verification. Microchannels with diameter variances of ±25 µm may be fine for prototyping but ± 10 µm is preferred for end-use devices.
Single-cell analysis MFD production requirements
PµSL meets all of these technical requirements for single-cell assay MFDs. It also offers the possibility of producing a complete MFD in a single build. If a user cannot print the complete MFD, individually printed components can be assembled using traditional methods. PµSL is also less expensive than precision machining or photolithography and can dramatically reduce development lead times while enabling more design iterations for reduced risk.
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A 15 × 7.2 × 2.7 mm MFD chip with 18 μm channels, produced on a microArch printer.
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A 15 × 7.2 × 2.7 mm MFD chip with 18 μm channels, produced on a microArch printer.
Digital PCR
Polymerase chain reaction (PCR), a microfluidics technique that rapidly makes millions to billions of copies of a target DNA sample, can also use MFDs that have been fabricated using PµSL. PCR amplifies the signal of a target gene and uses multiple thermal cycles across different temperatures. Digital PCR uses droplet microfluidics to provide an absolute DNA count so that a standard curve is not required. Droplet digital PCR (ddPCR) also provides greater sample partitioning for better statistics and requires only small sample volumes of fluid, an important consideration for rare or expensive samples. Microwells that withstand high temperatures are often needed for partitioning, but thermal cycling and/or measurement are not necessarily performed on the partitioning chip.
During digital PCR, a sample is portioned into tens of thousands of droplets. PCR cycles for all of these droplets are run simultaneously, and the fluorescence intensity of each droplet is measured. Based on this data, the concentration from a number of positive droplets is calculated. Applications for ddPCR include forensics, cellular biology, medicine, and the absolute quantification of viral load, including for SARS-COV-2. Digital PCR is also used in rare allele detection, genetically modified organism (GMO) detection and the enrichment and separation of mixtures.
PµSL technology can overcome the limitations associated with traditional manufacturing methods while meeting important technical requirements for digital PCR that are also common to single-cell assays. These include the ability to create hydrophilic channels with water contact angles of <30 and >150°, chemical compatibility with water-based and oil-based reagents, biocompatibility in terms of in vitro toxicity, and optical transparency of >80 percent transmittance.
Digital PCR MFD production requirements
MFDs that are produced with PµSL technology can also meet digital PCR requirements for channel diameters of 50 to 100 µm and microwell diameters of 50 to 60 µm. Moreover, there are available polymers that can meet microwell requirements for thermal dimensional stability up to 100 °C and low autofluorescene, the natural emission of light by biological structures.
Conclusion
PµSL technology strikes the right balance between precision and speed while providing application-specific benefits for fabricating the MFDs used with single-cell assays and digital PCR. BMF’s microArch 3D printers can produce small components with 2 μm resolution and +/-10 μm accuracy at scale. They also enable design complexity and support the use of resins that meet microfluidics requirements. BMF’s own UV-curable materials include acrylate-based resins that combine biocompatibility with high temperature and chemical resistance for microfluidics applications such as single-cell analysis and digital PCR.
Boston Micro Fabrication