Microfluidics is a growing field of research which pertains to the manipulation of fluids on the microscale level, and it is identified most commonly by devices with critical dimensions of less than 1 mm. At this scale, researchers can take advantage of the scaling of many physical laws and employ, for example, rapid diffusion [1
], laminar flows [2
], Dean flow [3
], rapid thermal transport [4
], and take advantage of the large surface area relative to the volume [5
]. These varied benefits have helped microfluidics find applications in many fields including analytical chemistry [4
], energy generation [6
], cell separations [3
], and molecular biology [7
]. As the field has progressed, many different methods have emerged for fabricating channels with the requisite dimensions.
Much of the work in the field of microfluidics was performed using soft lithography as introduced by Whitesides [8
] in 1998. The methods for soft lithography, especially in polydimethylsiloxane (PDMS), have been well documented [9
]. One of the principal difficulties of soft lithography originally was the requirement for cleanroom fabrication which, while well-developed by the microelectromechanical Systems (MEMS) community, remains costly and time-consuming. This has been overcome to some degree recently with low-cost mold making methods, as covered by Faustino [12
]. Microfluidic engineers have developed a variety of methods for fabricating submillimeter channels aside from soft lithography for various reasons, including decreased cost, faster turnaround time, cheaper materials and tools, and increased functionality. These developments have allowed microfluidic devices to be fabricated using a wide range of materials and geometries, enabling new and advantageous physical behaviors and qualities in microfluidic devices.
In this work, we provide an overview and comparison of several key fabrication techniques that are employed in microfluidic research today. These techniques include polymer laminates, 3D printing approaches, various polymer molding technologies, and recent developments in nanofabrication.
It has been our experience that starting a project with the correct fabrication technique can significantly accelerate a project’s timeline and improve the performance of the given device. This work specifically focuses on channel-based microfluidics, as opposed to paper-based devices. A helpful review of paper-based devices can be found [13
]. Further, this work also aims to cover those fabrication methods that do not require soft lithography. It builds upon other work such as the recent review by Walsh
] which offers a good review of fabrication techniques available for makerspace creators and has an emphasis on recent microfluidic applications and demonstrations.
Here we describe laminate microfluidic devices as chips, which are created as a stack of independently cut layers which are bonded together to form channels and microfluidic features. In these devices, each layer can be thought of in a two-dimensional flow geometry, which is closed by the layers above and below them. The height of a channel is then defined by the thickness of the material that is used to form the layer.
The most simplistic layered device is one composed of three layers: an interface layer, a flow layer, and a bottom layer [15
]. As an example, a three-layer device, used for microfluidic polymerase chain reaction PCR, is shown in Figure 1
. The device is fabricated by stacking a microscope slide, a flow layer cut from double-sided tape, and a microscope slide with ports cut into it, to form a closed channel [4
] with acrylic ports glued on as interfaces. This example illustrates many of the benefits of layered microfluidic devices in a laboratory/prototyping setting: relatively inexpensive materials and instruments, simple process steps, rapid fabrication times, well-controlled layer depths (set by material thickness), optical access, and submillimeter feature sizes. With the possible exception of 3D printing, layered devices are the fastest and easiest way to fabricate microfluidic channels for most applications. While fabrication is simple and rapid, available laboratory cutting methods generally limit feature widths to 50–200 µm, which may not be small enough for some microfluidic applications.
The four principal steps in forming a laminate device are (1) material selection, (2) cutting the desired microfluidic features in each layer, and (3) bonding the independent layers together to form one functioning device. Here, we provide a list of existing technologies in each of these areas with the intention of providing a resource for those hoping to fabricate such devices.
Laminate manufacturing methods are compatible with a very broad range of materials. Generally, a wide range of optically clear plastics and thermoplastics can be employed with laminates, as well as opaque layers, as desired. Three very common material categories in laminate device construction are adhesive transfer tapes [16
], polymer layers (commonly polycarbonate, PMMA, and COC) [17
], and glass slides. Adhesive transfer tapes are advantageous because the bonding is inherent in the material, although they may have the problem of chemical or particle absorption. Walsh et al. have compiled a valuable list of recommended adhesive transfer tapes for those looking to get started in the space [14
]. Polymers (including acrylic and polycarbonate) and glass layers are common for many of the same reasons: optical clarity, low cost, and sample compatibility. While these are the three most common materials, an immense number of other materials can be employed in laminate manufacturing. Indeed, due to their compatibility with so many different forms of bonding (including simple adhesives) laminate manufacturing is the fabrication method that is compatible with the widest variety of materials.
For laminate microfluidic devices, each layer is cut individually. The cutting method has a significant impact on the dimensions and the device functionality. For prototyping and laboratory settings, cutting is usually done with a knife plotter (i.e., xurography) or laser cutter because of the speed and simplicity that each tool offers. A knife plotter works by precisely cutting a material with a blade to create the geometry, while a laser cutter uses a focused beam (traditionally CO2
lasers are used). In both methods, the general process overview is the same: a device is designed using CAD software, the device geometry is cut using the selected method, the inner portion is cleaned (“weeding”), and the layers of the device are bonded together to form closed channels. In choosing between these two methods, the decision can be made based on the materials that will be employed, the thickness of the layers, and the dimensional accuracy required. A general review of this decision is available elsewhere [14
In general, laser cutters offer a better resolution (offering dimensional accuracy as small as 25 µm with optimized materials), and are more flexible in the materials and thicknesses with which they are able to work [18
]. However, they are in general much more costly (>USD $
10,000). For the laboratory and prototyping setting, Walsh, et al. offers a good review of the general benefits and costs associated with each cutting method [14
]. Besides the monetary cost of laser cutting, laser processing requires a vacuum pumping system, and it may leave burn residue that could impede the performance of the device. Cutting with a knife plotter is generally easier to set up than a laser cutter (it is less expensive and does not require a vacuum system), but less resolution is available (cutting features smaller than 500 µm can be challenging on some systems), materials and thicknesses are more limited, and more setup time may be required to determine the optimal parameters. Once running, the time required for cutting each piece is similar between a knife plotter and laser cutter. For many applications, the two cutting methods perform similarly, and a selection can be made based on the availability of instruments [16
Although the selection of the bonding method is inherently tied to the materials employed in the fabrication method, two of the most common and straightforward bonding methods are adhesives and thermal bonding. When employing adhesives as the bonding method, double-sided tape can be cut and employed directly as the flow layer or an adhesive layer can be applied between layers of different materials. This second method significantly increases the variety of materials that can be employed as adhesives are able to hold together most materials. While adhesive bonding is a significant enabling method, devices that employ adhesive bonding traditionally struggle to maintain high pressures (beyond 5 bar), and can be prone to uneven bonding and channel heights [19
]. In some cases, the pressure that can be sustained by adhesive-bonded devices can be increased. Demonstrated methods include plasma treating the surfaces before they are brought together, and the application of heat and pressure after the layers have been brought together [17
Thermal bonding is another straightforward bonding method which is commonly employed in microfluidic devices. To facilitate thermal bonding, the temperature of the individual layers is elevated to a temperature that is near the glass transition temperature of one or both of the materials, and a force is applied across the surface of the layers. When performed properly, the individual layers form one essentially indistinguishable solid material. While thermal bonding is not possible with all materials, it is particularly useful with polycarbonate and polylactic acid (PLA). The main disadvantage of thermal bonding is the possible warping of features from the heating or as the device cools down. Some laminating processes also leave bubbles between the layers. The compatibility of laminate microfluidics with thermal bonding often blurs the line between thermoplastic and laminate microfluidic manufacturing.
Since the layers of a microfluidic device are created one at a time, aligning the individual layers is an important preparation step for the bonding process. Many, if not all, device fabrication protocols include either an alignment step or an alignment tool used to prepare for this, with alignment holes of features being created in each of the individual layers in the cutting step.
The bonding method has a very significant impact on the pressure that the device is able to withstand, and this can significantly affect device performance. Poor bonding is also the source of many problems that plague layered devices, including the presence of air bubbles between layers (leading to leakage) and the deformation of features in the microfluidic device, which can seriously degrade device performance.
One of the primary benefits of laminate fabrication relative to other fabrication techniques is its scalability, especially when quantities exceed ~100,000 devices. Lamination is especially scalable because each individual fabrication step can be automated and run in a massively parallelized fashion. For example, as fabrication is pushed to commercial scales, individual layers could be cut through a stamping method. In a streamlined fabrication method, this cutting technique could prepare tens to thousands (UN6) of devices per minute. As device fabrication moves to this scale, fabricators could begin to take advantage of other modern microfluidic fabrication techniques. Such was the inspiration behind the concept of the Lab-on-a-foil fabrication, which employs and often blends thermoplastic and laminate based manufacturing methods to create devices characterized by cost-effective, high-volume fabrication, and self-contained disposable chips. Figure 2
shows what this fabrication method could look like at scale in a reel-to-reel processing scenario [20
]. As microfluidics move towards large scale commercial production, laminate based manufacturing will certainly play an important role in the development.
Though lacking the same dimensional precision offered by other microfabrication techniques, layered devices offer a simple platform for incorporating complexity by increasing the number of layers used in the device and by incorporating other functional and sensing layers (filters [21
], electrodes [22
], etc.). The incorporation of microfluidic chips with multiple flow layers is paramount when preparing a true lab-on-a-chip platform. In an early demonstration of the utility of laminate microfluidic chips, Weigl et al. created a hematology cartridge, shown in Figure 3
, which was used to process whole blood samples to determine red blood cell and platelet counts, hemoglobin concentration, and white cell differential count [23
]. By fabricating the chip across multiple layers, the authors are able to incorporate inlets, multiple storage areas, and a micro-cytometer on the chip. The authors noted that a minimum of five layers are required when channels pass over each other, which begins to illustrate both the large number of layers that may be required for some applications (e.g., a flow focusing element requires five layers), and as a corollary, the importance of proper alignment and bonding.
Nanostructures have been employed in many fields due to their unique properties, and they have found uses in microfluidics as well. As such, several fabrication techniques have been developed to generate 2D and 3D nanostructures. Nanofabrication techniques can typically be categorized into two groups: top-down methods and bottom-up methods.
Top-down techniques allow for good control of the size and distribution of features, and they commonly involve some form of photolithography. Photolithography involves the generation an outline of the desired micro/nanoscale device on a partially transparent photomask. After generation, an image of this mask is projected onto a substrate wafer that has been coated in a photosensitive polymer or “resist”. This enables the transfer of the generated pattern from the photomask to the substrate wafer. In standard photolithographic methods, an ultraviolet light source ranging from 250 to 435 nm is employed to create the image on the photosensitive material [78
]. While these sources can reliably produce micron sized features, the effects of diffraction prevent the resolution of sub-250 nm features onto the substrate. Due to diffraction, the limits of resolution of standard lithographic processes is typically on the order of the wavelength of light being used to create the image [78
]. Several techniques such as extreme ultraviolet lithography, electron beam lithography, and nanoimprint lithography have been developed to circumvent these limits. Lithographic approaches to reduce the feature size typically involve the reduction of the wavelength of the light source used to generate images onto the substrate. Subsequent deposition and etching steps follow until the device is complete.
While advanced photolithography techniques can provide sub-250 nm structures, the procedures required to produce such structures are highly serialized and slow. Pattern transfer via lithographic methods is also often subject to defects due to defects on the mask, contact with the mask, and diffraction of light. Finally, use of lithography techniques is expensive since these techniques often involve extensive use of clean room equipment. There has recently been a desire from many different sectors of industry for the rapid fabrication of periodic and ordered nanostructures. Bottom-up nanofabrication techniques have been proposed as an alternative to circumvent the shortcomings of photolithography. Bottom-up nanofabrication techniques involve the formation of nanoscale structures through the self-arrangement of atomic and molecular building blocks. This molecular nature allows for highly repeatable, periodic structures.
In this review, three nanoscale lithographic methods (Extreme Ultraviolet, Electron Beam, and Nanoimprint lithography), as well as one non-lithographic (Anodic Aluminum Oxidation), are examined in relation to microfluidic fabrication. The working principles, applications to microfluidics, and prospects for commercialization for each technique are evaluated.
5.1. Extreme Ultraviolet Lithography
Extreme ultraviolet lithography (EUV) is an optical lithographic process like standard lithography described above. However, EUV uses 13 nm light for substrate exposure [80
]. To generate these small wavelengths of light, a laser is used to illuminate a plasma, such as a xenon plasma. The plasma, in turn, emits the EUV light. This light is concentrated using a system of mirrors which have up to 70% reflectivity of light. The EUV light is then shone from a reflective mask into a system of reducing mirrors. These mirrors reduce the spot size of the light beam into the desired double-digit nanometer range. This light is then shone onto a special photosensitive resist, after which typical developing and etching protocols can be followed [81
While reports of fabrication of microfluidic devices with the use of EUV are rare, extreme ultraviolet lithography can be used for the fabrication of nanopillars to study cells. Both the use of nanopillars to study cells in microfluidic devices and the use of EUV to fabricate pillars have been reported previously [82
]. Additionally, EUV has been found to be advantageous over other standard lithographic techniques for the creation of microfluidic and nanofluidic channels with varying geometries when it is combined with gray-scale lithography [84
Otherwise, it seems that EUV applications in nanofluidics have been limited, but are likely to develop as research moves to smaller length scales. However, due to its high cost, it is unlikely that EUV will see extensive applications in microfluidics.
Commercially, however, extreme ultraviolet lithography has found its niche in the semiconductor industry. Practical EUV use has been reported in the creation of integrated circuit chips. Recently, IBM reported the creation of 5 and 7 nm transistor nodes using extreme ultraviolet lithography. The 5 nm chip offers a 40 percent increase in performance, while using 75 percent less power than the 10 nm chips that are currently on the market. While the 5 nm chips are still in the developmental stage, the 7 nm chips will be available on the market in 2018 [85
]. Companies like IBM and Intel will continue to provide funding for the development of EUV as they view it as the best prospective technology to reduce the size of transistor nodes [86
5.2. Electron Beam Lithography
Electron beam lithography (EBL) is a lithographic method derived from the development of the scanning electron microscope (SEM) [87
]. Like other lithographic processes, its focus is the transfer of patterns onto a structure using a reactive resist. However, unlike other lithographic methods, it uses a focused electron beam, rather than light, to expose an electron reactive resist. When the high energy electron beam interacts with the resist coating, either the solubility of the resist is increased, or a crosslinking of polymers occur. EBL is a mask-less lithographic technique, since the electron beam that is used to expose the surface is rastered over the surface to generate the patterns that are desired. This makes it advantageous over masked techniques, since defects due to masking are no longer present. However, the rastering process of EBL makes it a fairly low throughput process [88
]. Electron beam lithography is advantageous over conventional lithography because of the extremely small feature size generation possible. Since EBL does not involve the typical optical elements used in standard photolithography, it is able to overcome the diffraction limited minimum feature size of standard photolithography, and it has been able to generate feature sizes less than 10 nm [78
While rare, the use of electron beam lithography for the manufacturing of microfluidic structures has been proposed [89
]. Electron beam lithography can be used in micro- and nanofluidics for the creation of nanochannels. It is also an ideal technique for use with tissue engineering and biosensors [88
]. Moolman et al. reported the fabrication of a microfluidic chip, with nanochannels ranging from 300 to 800 nm in width and 1.2 µm in depth, with the use of EBL. This device was used to study the growth of several submicron species of bacteria over many generations. Channels created on a silicon wafer were used to create a stiff negative PDMS master mold, which in turn was used to create several softer PDMS channels. The final PDMS channels were bonded to a glass cover-slip. Figure 8
summarizes the fabrication process for this device. Moolman proved the functionality of the fabricated device, by growing both Escherichia coli
and Lactococcus lactis
in the fabricated devices for several generations. The use of EBL in this work provided Moolman et al. greater control of the size and shape of structures in their microfluidics device when compared to a similar device that was constructed using conventional lithographic techniques [90
Given that EBL originates from the modification of already existing technologies, its adoption into spaces where SEMs are already present should not be too laborious. As such, commercial production of electron beam lithography tools is ongoing, and companies are actively developing new and improved EBL systems, though they remain expensive and slow [92
]. However, due to its highly serial nature, EBL will likely not be readily adopted in fields requiring the high throughput creation of devices such as microfluidics.
5.3. Nanoimprint Lithography
While E-beam and EUV lithography do satisfy the need for sub-20 nm nanostructures, they are both fairly expensive and low throughput processes [93
]. There is, therefore, a desire for high throughput, low-cost lithographic processes that can easily generate sub-20 nm structures in high-throughput and at a reasonable cost. Nanoimprint lithography (NIL) is such a process. Like stamping, nanoimprint lithography is primarily a mechanical process in which a prefabricated mold is pressed into a resist material. After the mold is pressed into the material, further hardening of the resist can be achieved using either thermal, chemical, or optical methods such as UV curing. After sufficient deformation of the resist material has been achieved, the mold is removed from the resist surface and further development steps can take place [94
As it is a low cost, high throughput technology, NIL has been applied for the fabrication of many different microfluidic devices. Isobe et al. built a microfluidic system which consisted of micrometer channels as well as an array of parallel 500 nm wide and 1–2 µm tall channels for delivering solutions in microfluidic bioassays. To achieve this, a combination of UV-assisted thermal NIL, as well as standard UV lithography, was used. SU-8 was spin-coated on a silicon wafer and pre-exposed to initiate crosslinking in the SU-8. Following this, thermal nanoimprint lithography was performed to generate the nanochannel array. After post exposure, an additional layer of SU-8 was spun onto the wafer and processed using standard UV lithographic processes. The SU-8 structures generated by these processes were used as a mold to form channels in PDMS [93
]. Figure 9
provides a schematic of this device.
Nanoimprint lithography displays promise for wide commercialization. NIL is both a high throughput and a low-cost process. These features are very desirable to the semiconductor and microfluidics industry. Due to its advantages over extreme UV and E beam lithographies, high profile companies like Canon have begun to make large investments in its advancement [95
]. Advancements in NIL, such as roll-to-roll NIL, promise to improve the throughput of NIL even further, though at the cost of minimum feature size [96
]. Recently, roll-to-roll NIL was used to continuously imprint a polyethersulfone ultrafiltration membrane onto a substrate [97
]. This illustrates NIL’s application in the fabrication of commercial devices, both in microfluidics and in the semiconductor industry.
5.4. Anodic Aluminum Oxidation
The use of oxidized aluminum to prevent wear and corrosion has been commonplace for a long time [98
], but it has been found that these structures are also useful for membranes and nanofluidics. Anodic aluminum oxidation (AAO) occurs when aluminum is placed in an acidic electrolyte and current is passed through the solution with the aluminum sheet placed on the anode, resulting in the formation of close-packed, uniform, porous hexagonal cells. These cells can range in diameter from 4 nm to 200 nm, and can grow perpendicularly to a height determined by the electrochemical conditions. Techniques for the fabrication of highly ordered metal nanohole arrays, such as gold nanoholes, based on AAO have developed, and they are reported by Masuda and Fukuda [99
]. To achieve these nanoholes, first, a porous aluminum oxide layer is developed on an aluminum substrate at appropriated anodization voltages. Next, a thin metal layer is deposited on top of the porous aluminum with the use of physical or chemical vapor deposition techniques. Following this, a resist is used to fill up the porous alumina and developed. This step creates a negative cylindrical resist mold that may be used to create further nanoholes with methods varying from soft lithography to metal plating [100
Current research on the fabrication of nanostructures using AAO involves the use of hierarchically branched AAO templated to fabricate asymmetric nanopillars [101
]. While the reported use of AAO for microfluidics is limited, potential applications still exist. The use of AAO for the formation of ultrathin membranes has been widely reported [102
]. Recently, Sharma and Gale reported the integration of an aluminum oxide membrane (AOM) into a microfluidic device, and its use for the electrochemical quantification of DNA [103
]. Given that PDMS, a primary polymer of choice for microfluidics, does not bond well with AAO membranes, the aluminum oxide membrane in this case had to be embedded in double-sided tape. Figure 10
shows a schematic of the manufactured device. This device was able to successfully capture genomic DNA due to the charge on the AOM. Additionally, the use of AAO membranes has been reported for the development of highly organized arrays for use with microfluidics. Chen at al. reported the use of an AAO membrane to organize and dry an Ag nanodot array onto the surface of a quartz slide. This slide was then sealed within a microfluidic channel made of PDMS, and the device was used to perform surface-enhanced Raman spectroscopy [105
The anodic oxidation of aluminum stands a good chance of becoming a standard process in microfluidics. Given that commercial AAO has existed for a long time in other industries, the equipment with which AAO can be performed is readily available. It is also a rapid technique in comparison to the fabrication of nanopillars/pore with methods like extreme ultraviolet lithography. AAO membranes are already made by companies like GE Whatman. The potential for AAO to generate complex nanopillars arrays that can be used in cell biology and diagnostics make it a worthwhile nanofabrication technique that should garner more attention than it currently receives.
Newly developed manufacturing techniques that go beyond soft lithography have helped to bring microfluidics into regular use by academia, industry, and the public. The techniques presented in this review help provide insight into the current and upcoming manufacturing processes that will important in microfluidics.
Molding has been the fundamental process used in academic microfluidics labs for many years. The approach of replica molding using PDMS continues to provide a valuable platform for research, prototyping, and custom biomedical microfluidics, while injection molding and hot embossing are popular in producing commercial microfluidic devices. The recent advances in replica molding appear to be partially focused on providing improved tools for 3D cell culturing. Research in injection molding and hot embossing aims to address limitations in mold cost, as well as further improve the functionality of existing methods and materials.
The use of laminates, undoubtedly, has a long-term future in microfluidics, and it is especially practical in moving microfluidics toward commercialization. Perhaps we will see 3D printed devices encroach on space traditionally claimed by laminates, but laminates perform far better in scaled-up manufacturing and enable the incorporation of multiple materials and components. These benefits across multiple materials and with an increasingly broad range of materials will become even more apparent, especially when recognizing recent advancements in bonding and alignment methods.
The use of nanoscale structures in microfluidics is widespread for the study of bacteria growth, cell adhesion, detection of biomarkers, and the study of diffusion. For a nanofabrication method to be successfully adopted for use in microfluidics, it is required that the process be low cost, high throughput, generate small feature size, and reproducible. While many of the methods mentioned can reproducibly create sub-20 nm structures, fabrication methods such as electron beam lithography and extreme ultraviolet lithography currently have their use in microfluidics is limited by both their cost and serial processing. High throughput methods like nanoimprint lithography and anodic aluminum oxidation, however, are likely to generate more use in microfluidics. Both methods meet the requirements for rapid prototyping, have seen some commercial success, and can be performed with simple equipment.
There have been some barriers to commercializing microfluidic devices that traditional fabrication methods have failed to address, but that may be solved by 3D printing. Specifically, there are three barriers that need to be overcome: non-standard user interfaces, complex control systems, and the fact that liquid polymer (i.e., PDMS) molding is not easily commercialized due to speed and cost concerns. In many instances, 3D printing seems to be the solution to these problems. It is inexpensive for making prototypes, and the technologies are getting more cost-effective and faster each year. A variety of materials have been developed to meet the need of microfluidics, with properties such as being transparent, non-fluorescent and biocompatible. However, 3D printing has not been able to produce the same resolutions as PDMS manufacturing, in its current state. There have been great improvements in 3D printing of finer channels in recent years using SL, as well as 3D printing using two-photon manufacturing to achieve submicron resolution., though two-photon polymerization does tend to be expensive and slow. Biocompatibility does need to be addressed with processes such as SL and MJM, though hydrogels such as PEGDA have shown promise in this area. As 3D printing improves, advances in 3D printing with different materials and resolutions have the potential to develop integrated microfluidics devices in a single step process with multi-material capabilities and integrated circuits [106
] 3D printing has had great improvements due to its recent popularity, and it has the potential to have a lasting impact on the field. There is a high probability that as 3D printing improves, it will replace nearly all the other methods of microfluidic device manufacturing in research and a good probability that 3D printing will become the primary commercial manufacturing methods due to its straightforward scale-up and custom device manufacturing potential. Overall, multiple methods for manufacturing microfluidic and nanofluidic devices exist, and rapid development of these methods is allowing for new and unique applications in the medical, biological, and chemical areas.