MOLDING - ONLINE EXPANDED VERSION
A fluidic card is constructed from layers of acrylic bonded with an optically clear bottom and a gas-permeable top. A single inlet distributes nutrients evenly across all 10 sample chambers.
In the development of molecular and immunodiagnostic products, many mechanical components need to come together and perform as a unit. Because the components are engineered and modeled individually, unexpected behaviors often arise in the performance of these devices. When a device incorporates microfabricated components that may be injection molded or embossed, changes in the design need to be carefully considered because they can lead to considerable expense in retooling. To avoid these costs, modifications are made reluctantly, and other work-arounds may be proposed. Although such fixes lower short-term development costs, the result is often a merely adequate, but not superior, product. However, early prototyping and development offers an alternative to the expense involved in tooling for injection-molded microfabricated components.
What Is Polymer Laminate Technology?
Polymer laminate technology is a microfabrication method for rapid prototyping and volume manufacture of biological microfluidic devices. These devices are used for applications such as spectroscopy for environmental analysis, sample preparation, kinetic assays, microfluidic-based molecular and immunodiagnostic devices, and automated cell culture. Manufacturing of these devices begins with thin sheets or films of a variety of polymeric materials to form multiple stacked layers of fluidic channels and vias that are bonded (laminated) together with a pressure-sensitive adhesive or thermal bond to form a complex fluidic unit. Channels and other functional structures are precision laser cut in the layers of polymer substrate, as well as in the bonding adhesive. Lamination technology and equipment are already a part of this process. They can be used in new ways to cut expenses because it is not necessary to invest in new manufacturing technologies.
Fabrication at the Microscale
Traditional fabrication technologies, such as injection molding, embossing in plastic, and etching in silicon, are expensive to develop and risky in molecular and immunodiagnostic product development for a couple of reasons. They are economical to manufacture only at very high volumes (millions of devices per year) and have no track record in this particular market.
Diagnostic tests are shifting from single, highly complex tests (that are performed in traditional diagnostic labs and take two to three days to render results) to near-patient panels that provide results in mere minutes to hours. Therefore, many of the operations that were once performed by large robotic systems or by hand in a test lab are increasingly being integrated into devices that combine sample preparation with assay incubation and final measurement of the result. Such devices are too small and sophisticated to be manufactured using traditional methods.
Flexibility in Material and Structures. Polymer laminate technology has several advantages in fabricating diagnostic tests. Traditional fabrication approaches do not allow the incorporation of high feature densities, 3-D structures, or integrated functionality that are associated with self-contained tests. Polymer laminate fabrication allows the design of complex functionality, such as flexible pneumatic valves, recirculating pumps, filtration components, and simple interfaces to optical or electroactive components. A wide choice of materials with essential performance characteristics (e.g., polymethylmethacrylate (acrylic) cast or extruded, polyethylene terephthalate, polystyrene, polypropylene, and medical-grade silicone sheets) means lower development costs and rapid, inexpensive modifications to the design. Because all polymer laminate fabrication processes are room temperature, reagents may also be incorporated and will not be damaged.
It is a common misconception that the walls of integrated channels cannot be made smooth or that the adhesive extends into the channel, which may cause reagents to hang up during the course of an assay, making laminates unsuitable in these applications. However, the high quality of the bonding adhesives provides edges as smooth as the edges of the substrate materials that are cut, with no running or bulging of the adhesive into the channel. Experience has shown that cell culture, immunoassays, and polymerase chain reaction (PCR) are all compatible with laminate devices fabricated using select adhesives.
In applications for which the devices have been used with multiple wash steps, the clearance of the channels was within the expected five volumes calculated from fluid modeling. In many applications, particularly the common single use, carryover or hold-up volume is easily compensated for by providing a small overvolume. Problems with nonspecific binding no worse with the presence of the adhesive, which in all cases accounts for less than 10% of the total exposed surface area that the liquid contacts. With a choice of buffer and additives, enzyme activity can be preserved and PCR run.
Precision Registration of Fine Features. To accomplish repeatable performance in microfabricated devices requires attention to robust and repeatable alignment of the layers that are stacked and bonded to create the 3-D fluidic network. This calls for stacking and alignment tolerances of 50–125 µm with feature sizes in the device of typically 100–1000 µm. Alignment is achieved using fixtures with alignment pins that mate with features in the device. Stacks of 13 layers have been made with alignment tolerances of 50 µm throughout the stack. Typically, alignment requirements can be relaxed by oversizing features at the point where they connect to another layer.
Polymer laminates made using thick acrylic material perform well as manifolds.
Reduced Device Footprint and Cost. The ability to form enclosed 3-D structures, such as channels or chambers, without a separate bonding process, reduces the device’s footprint and therefore its cost. Further, it makes devices highly compatible with the work flow in a hospital laboratory setting. A panel can be run on a single small chip rather than having separate chips for each test. The ability to stack channels and functions in the vertical dimension facilitates the integration of all the required steps.
To accomplish the same functionality with traditional injection molding would limit the device to three layers, in which functionality can reside on one of two sides of the device. With polymer laminate fabrication, the channels and vias that connect them can be stacked up to 13 layers or more, permitting a compact, low, dead-volume system to be developed.
Rapid Prototyping. Polymer laminate technology is suitable for rapid prototyping and enables empirical testing. For instance, when it is necessary to use an experimental design to understand the performance of a device, changes in the polymer laminate channel geometry are simple to make to see how each change affects the assay. Channel dimensions can also be easily adjusted, which would not be possible with an injection-molded part. Therefore, a range of modifications to the design can be evaluated in parallel, allowing experimental design to enter into the development. In addition, a single fabrication run can incorporate a variety of different modifications to avoid the need for multiple runs to test different designs.
Customization of Diagnostic Platforms. Typically a diagnostic platform is developed for use in multiple tests because it is not cost-effective to manufacture an entirely new test platform for each diagnostic test. To use the same platform, it must be customized for different incubation times and reagents and often for different materials. Polymer laminate technology makes this customization possible without a large investment in tooling.
Robust Adhesion. There is a common misconception about the quality and performance of the adhesive in this new technology. Device manufacturers sometimes fear that the adhesives between polymer substrates will lack structural integrity and will interfere with the device’s performance. However, the adhesives used in polymer laminate manufacturing are the strong, biocompatible silicone or acrylic-based, pressure-sensitive adhesives that are typically used in the medical device market. They are optically clear and provide, upon complete cure in 12–24 hours, a permanent bond to the substrate. In fact, the bond can be so strong that the material that brings the two pieces together would break apart before the adhesive would break down.
Alternatives to pressure-sensitive adhesives include a 25 µm polypropylene thermal bond adhesive (melt temperature of 160°F) and bond materials that work through van der Waals interactions with the substrate surface as the polypropylene wets the surface.
Interface with Traditional Components. Laminate components can serve as a sort of functional sticky tape to mate with injection-molded components and interface with electro¬active or optical detection components. The laminate component cost-effectively brings together pieces of the puzzle that could not otherwise be combined. Thus, polymer laminate technology is an important step in bringing microfabrication technologies more fully into the market.
Easy to Scale. Further efficiencies are achieved from a linear scale-up process that facilitates volume manufacture. Because the process is linearly scalable, increasing manufacture from 1000 to 100,000 per month can meet the needs of device developers that are just launching a product. Without a large initial investment in manufacturing capability, they can grow and begin generating revenue that will ultimately support an investment in long-term and high-volume manufacturing.
Rapid Turnaround Time. The high feature density of multiplexed diagnostic devices means not only more-expensive tooling, but also a long turnaround time, as much as six weeks, to receive parts for testing. With polymer laminate technology, development time is drastically reduced and initial prototypes can be available within three to seven days.
Although polymer laminate technology is robust, proven and inexpensive, it is not a panacea. It is not cost-effective for fabricating millions of devices per year for multilayer laminates. A manufacturer that produces devices in such large quantities can afford to invest in expensive tooling and reengineering of traditional methodologies. The strength of polymer laminate technology is in applications of single-use, disposable devices that need to accomplish many different tasks. The device takes the complexity of the technology out of human hands and puts it into a small fluid card.
Applications of Polymer Laminate Technology
A five-layer laminate component for a molecular diagnostic device that tests susceptibility to warfarin. The laminate brings an injection-molded component for sample introduction and a PCB together. The laminate functions as a recirculating pump to enhance target DNA capture.
Example 1: Electrochemical Detection-based DNA Microarrays. Osmetech Molecular Diagnostics (Pasadena, CA) is in the final stages of obtaining FDA clearance for a DNA microarray device that incorporates polymer laminate technology. The eSensor XT-8 is a DNA microarray device for warfarin sensitivity testing. Warfarin, one of the most commonly prescribed anticoagulants in the United States, exhibits a narrow therapeutic range, a wide interindividual variation in dosage required to reach optimal therapeutic effect, and severe adverse effects from overdosage due to excessive bleeding.
The eSensor XT-8 cartridge device establishes an individual’s genotype. It consists of a PCB chip, a cover, a plate, and a microfluidic component composed of a laser-cut multilayer laminate. The microfluidic component includes a diaphragm pump and check valves in line with a serpentine channel that forms the hybridization chamber above the array of electrodes. The printed circuit board (PCB) chip is prepared for an eSensor assay by depositing DNA capture probes and insulator molecules on the working electrodes. Each specific deposition solution is dispensed on the appropriate electrode using a robotic pipette system.
The capture probe and insulator react with the gold surface to form an insulating self-assembled monolayer. After capture-probe dispensing, the PCB chips are washed, dried, and assembled with the laminate, plate, and plastic cover into a cartridge to form a microfluidic circulating system that can hold approximately 140 µl.
Osmetech is one of the few companies to provide electrochemical-based detection technology for nucleic acid analysis. Conventional microarray devices are expensive and they rely on fluorescent detection-based technologies that require bulky optical detectors. Polymer laminate is low cost and also has a small footprint, which conserves bench space in a typical clinical laboratory.
According to the company, previous studies demonstrated the feasibility of a single-use, sample-to-answer eSensor device, and recent developments in microfluidics provide additional tools to perform the required functions. Point-of-care microarray systems using electrochemical detection of nucleic acids can meet critical healthcare needs, including rapid genotyping.1
Example 2: Particle Sorter. CFD Research Corp. (CFDRC) conducts high-end R&D for government agencies and other organizations. The company uses computational tools for component and system design, gives those data to the manufacturers for fabrication, and then performs testing with the completed devices. One of the CFDRC’s products that incorporates polymer laminates is a dielectrophoresis (DEP)-based particle sorter. The DEP sorter is designed to allow intelligent discrimination of target particles from background clutter. The sorter can be used to effect separation based on size (respirability) or particle type (dielectric properties). It can operate in batch, stopped-flow, or continuous-flow modes.
Polymer laminate technology is uniquely suited for the DEP sorter because of the two-sided nature of the device. The device has a metal layer on the bottom, followed by a polymer laminate layer that contains the fluidic channels, topped with another metal layer, all bonded together with pressure-sensitive adhesives.
Microsystems such as these have very small channels that call for microfabrication. The channel in which the sample is introduced is about 100 × 500 µm, which is difficult to manufacture using traditional machining methods. An alternative approach is traditional lithography, but two-sided lithography is not very well established. Substrates such as glass or silicone could be used, but plastics are often more cost-effective for technology demonstration. Glass is more robust and user-friendly for working with biologicals, and might be a better choice in the long run. However, if suitable coatings can be added, the lower cost associated with plastics would cause them to be more readily adopted by the market.
Another advantage of laminate-based technology is the ability to stack multiple layers and attach them using a pressure-sensitive adhesive. For a silicon or glass substrate, it would be necessary to use anodic or fusion bonding with either high temperature, high voltage, or both to attach the top and bottom layers. Both bonding processes are cumbersome (especially glass-glass bonding), and they are permanent. The adhesive that bonds laminate layers, on the other hand, can be dissolved so that the layers can be reused. Instead of manufacturing 100 devices, you can make 20 and reuse them five times. The reusability combined with lower cost makes plastics more attractive than either silicone or glass at the proof-of-concept stage.
CFDRC’s next step is to develop an integrated sample-preparation cartridge for processing complex liquid samples to test for and identify pathogens. It will combine multiple components on a single chip that would interface with off-chip controllers to carry out multiple steps in an automated fashion. The components are modular by design and may be combined to yield an integrated sample-prep unit. For example, the particle sorter may be combined with a device that uses an electric field to extract the intracellular content from the bacteria and have it analyzed by a sensing element. The resulting product would be a handheld sensor for biological analysis.
Example 3: Rapid Prototyping for Fluidic Device. Blood Cell Storage Inc. has submitted a new fluidic device for patent review and is in the process of prototyping it using polymer laminate technology. Once the prototypes have been proven and the design finalized, the product will scale up to high-volume production. The company plans to outsource this phase. Because polymer laminate technology is flexible, the turnaround on the prototypes is very fast. The design can be altered and a new prototype batch of 25 parts delivered within two or three days.
Laminated construction is particularly suited to this design because it provides the ability to combine layers of dissimilar materials, such as plastic and glass, which are critical to the device. The company can submit various designs and test them much more quickly than it could using a different fabrication technique. Polymer laminate technology also provides very consistent results. The firm can place an order for 100 parts with delivery expected in less than two weeks. In the past, Blood Cell Storage Inc. built prototypes in-house but lacked the capability to achieve the high-quality bond between the layers. Outsourcing the construction of prototypes saves the firm time. It is also cost-effective because the outsourcer is geared up to laminate on a large scale and therefore can provide an ample supply of matching parts quickly.
Branched channel structure showing onboard, keyhole-shaped pneumatic valves. The device is used for multiplexed analysis and integrates with an injection-molded structure containing the reservoirs.
Example 4: Fluidic Card in Nanosatellite for Biological Experiments in Space. NASA’s Gene-Sat 1 Technology Demonstration Mission is a fully automated, miniature space flight system that provides life support for small living things and conducts biological examinations to look for genetic changes in bacteria during spaceflight. Putting novel cellular genetics investigative tools into small spacecraft gives scientists the ability to study and understand the effects of deep space environments on living things. The Gene-Sat 1 experimental payload was designed to perform assays of biological specimens in an automated fashion.
One of its subsystems is a fluidic card that features one inlet fluidic channel and one outlet fluidic channel that feeds 10 sample wells at the same time. The device was constructed using layers of close-tolerance cast acrylic bonded with an acrylic-based pressure-sensitive adhesive. The inlet and outlet to each well has a 0.45-µm porosity nylon membrane which serves two functions: to keep the suspended cell culture from moving between wells, and to provide even flow of the nutrient solution across all 10 cell culture chambers by having equal membrane area at the inlet and outlet of each culture chamber. The top-most layer of the device consists of a gas-permeable membrane, also bonded using a pressure-sensitive adhesive.
The technology demonstration satellite was launched in December 2006 containing 10 experimental samples of E. coli that were genetically modified to produce a green fluorescent protein during growth. Once the satellite was in orbit, autonomous incubation of the E. coli cultures was initiated by an increase in temperature and the infusion of a nutrient solution to each of the membrane-isolated cell culture wells built into the laminated microfluidic cell culture card. The bacterial growth was monitored for 96 hours by miniaturized optical back scattering and fluorescence detection positioned to observe both sides of each of the cell culture wells through the optical and UV transparent polymer walls. Each of the 10 bacterial cell cultures grew and successfully expressed the fluorescence signature of their special genetic marker. Data from the fluorescent E. coli was telemetered back to earth for analysis.
Upon reviewing the mission science data, the researchers found it successful. The organisms grew and glowed, and the sensors all worked. The ability to incorporate the porous membranes proved to be critical to the success of the device’s performance for this mission. The laminate construction proved to be robust and to meet the rigorous demands of the launch environment.2
New Products to Market
Polymer laminate technology complements, but does not replace, injection molding. It adds value to the manufacture of up to a million devices per year and facilitates the development of devices that might not otherwise reach the market due to tooling costs. It lowers the barrier to entry for device manufacturers with groundbreaking ideas for diagnostic tools and others who want to address smaller markets. By enabling a shorter development time and the production of multiple iterations inexpensively, polymer laminate technology speeds time to market and makes the microfabrication of devices with complex features affordable.
Leanna Levine , PhD, is founder of ALine Inc. (Redondo Beach, CA). Contact her at firstname.lastname@example.org.
1. Robin Liu et al., “Electrochemical Detection-Based DNA Microarrays,” In Vitro Diagnostics Technology 14, no. 2 (2008).
2. Antonio Ricco et al., “Autonomous Genetic Analysis System to Study Space Effects on Microorganisms: Results from Orbit,” Transducers and Eurosensors 2007: The 14th International Conference on Solid-State Sensors, Actuators, and Microsystems, Lyon, France, June 10–14, 2007.