High-volume processes for next-generation biotechnology devices

BY DR. BERND DIELACHER, DR. MARTIN EIBELHUBER and DR. THOMAS UHRMANN, EV Group, St. Florian, Austria

Over the past several decades, miniaturization has significantly improved clinical diagnostics, pharmaceutical research and analytical chemistry. Modern biotechnology devices— such as biosensors, fully integrated systems for diagnostics, cell-analysis or drug discovery—are often chip-based and rely on close interaction of biological substances at the micro- and nanoscale. Thus, process technologies that enable the production of surface patterns and integration of fluidic components with small feature sizes are needed (FIGURE 1).

FIGURE 1. Biotechnology devices utilize a variety of structures at the micro- and nanoscale that interact with biological substances.

FIGURE 1. Biotechnology devices utilize a variety of structures at the micro- and nanoscale that interact with
biological substances.

Today’s miniaturized biotechnology devices can be found in numerous applications, including fields related to human health as well as environmental and industrial sciences. For example, chemical sensors and biosensors are commonly used to analyze pH values, levels of electrolytes and blood-gas. Glucose sensors are a prominent example of highly successful commercial devices used for diabetes monitoring, where miniaturization has enhanced the development of implantable chips for continuous glucose level monitoring inside the human body.Fully integrated systems, including micro- and nanopumps for accurate insulin release, have also been shown. In general, such controlled drug delivery systems offer new opportunities for the treatment of common acute and chronic diseases. Moreover, microneedle arrays, which allow minimally invasive and painless delivery of drugs through the skin, neural electrodes for stimulation or monitoring signals inside the brain, or prosthetic devices such as artificial retinas, have also been developed.

Microfluidics plays a key role in the transport and manipulation of biological fluids in biotechnology devices. For example, laminar flow behavior can be overserved, which allows a well-defined control of liquids. Capillary forces can enable fluid flow without the need of active pumps. In addition, short distances reduce diffusion times of molecules, which lead to faster biological reactions. Overall, microfluidic devices offer a high degree of parallelization while using extremely-low-volume samples. Microfluidic devices that perform complete tasks or analysis, usually done in a laboratory, are referred to as lab-on-chip (LOC) devices. Other names include bio-chips or micro-Total-Analysis- Systems (μ-TAS). These systems are used in applications such as in-vitro diagnostics, high-throughput screening, genomics and drug discovery. LOC devices are also ideally suited for point-of-care testing (POCT), where they provide rapid diagnostics at the patient site.

Nanoimprint lithography

To successfully commercialize such products in a fast growing market with stringent requirements and high regulatory hurdles, precise and cost-effective micro- structuring technologies are essential. Nanoimprint lithography (NIL) has evolved from a niche technology to a powerful high-volume manufacturing method that is able to serve today’s needs and overcome the challenges of increasing complexity of microfluidic devices in particular, and biotechnology devices in general. NIL is a patterning technique capable of producing a multitude of different sizes and shapes on a large scale by imprinting either into a biocompatible resist or directly into the bulk material with resolutions down to 20 nm. NIL can be distinguished between three types of imprint technologies: hot-embossing or thermal NIL, UV-NIL, and micro- contact printing (μ-CP).

Hot-embossing is a cost-effective and relatively simple process, well suited for the fabrication of polymer microfluidic devices with very high replication accuracy of small features down to 50 nm (FIGURE 2). A polymer sheet or spin-on-polymer is heated above its glass transition temperature, transforming the material into a viscous state. A stamp containing the negative copy of the struc- tures is then pressed into the polymer with sufficient force to conformally mold the polymer. De-embossing is done after cooling the substrate below a certain temperature where the material retains its shape when removing the stamp. During hot-embossing, the structure is trans- ferred by displacement of the viscous material. The process is characterized by short flow paths of the material, moderate flow velocities and imprinting temperatures. Residual stress is therefore low, especially when comparing the process to injection molding, which is an alternative production technique for microfluidics.

FIGURE 2: a) 200-μm wide microfluidic channels and b) 10- μm pillar arrays with high aspect ratios (7:1) fabricated by hot- embossing (Courtesy of National Research Council Canada). c) Schematic drawing of hot-embossing process flow.

FIGURE 2: a) 200-μm wide microfluidic channels and b) 10- μm pillar arrays with high aspect ratios (7:1) fabricated by hot- embossing (Courtesy of National Research Council Canada). c) Schematic drawing of hot-embossing process flow.

FIGURE 2: a) 200-μm wide microfluidic channels and b) 10- μm pillar arrays with high aspect ratios (7:1) fabricated by hot- embossing (Courtesy of National Research Council Canada). c) Schematic drawing of hot-embossing process flow.

Because of the much higher process temperatures and pressures associated with injection molding, final products produced by this process usually experience higher internal residual stress, which easily results in significant deformation, such as warpage and shrinkage. In addition, a surface solidifi- cation layer is formed at the interface of the cold mold during the injection of the high-temperature molten polymer. This effect significantly influences the replication accuracy and optical quality. Extensive effort in process development and simulation is therefore often necessary for injection molding to replicate small features in an accurate manner. In contrast, hot-embossing allows precise replication of micro- and nanostructures with less effort and is superior when replicating high-aspect ratio features or when using very-thin substrates. Structures with high-aspect ratios are often needed in microfluidic chips for filtration elements, particle separation or cell sorting.

The ability to use very thin substrates enables the patterning of spin-on-polymer layers on top of other materials or even roll-to-roll embossing using polymer foils for very-high-throughput production. Parallel wafer-based batch processing also enables fabrication of typical-sized microfluidic chips with throughputs compa- rable to or even higher than injection molding or similar techniques. Since master stamps for hot-embossing do not need to withstand the high temperatures and forces required for mold inserts for injection molding, they are less expensive to produce. Therefore, hot-embossing is also a well suited technology for R&D and allows easier design changes in volume-production. UV-NIL refers to a technique where a transparent stamp is pressed into a photo-curable resist and cross-linked by UV-light while still in contact (FIGURE 3). In biotechnology applications, the resist is usually coated onto silicon or glass substrates. Unlike hot-embossing, the UV-NIL stamp is brought in contact with the resist using minimum force to conformally join the stamp and substrate. The different mechanisms of curing and stamp attachment account for different advantages and fields of application of the respective technologies.

FIGURE 3: (a) 100-nm grating with residual layer <10 nm imprinted into 90 nm height resist on silicon substrate and (b) 350-nm photonic crystal fabricated by UV-NIL. c) Schematic drawing of UV-NIL process flow.

FIGURE 3: (a) 100-nm grating with residual layer

UV-NIL provides very-high-alignment accuracy, pattern fidelity, and throughput whereas hot-embossing is capable of imprinting higher aspect ratios and larger structures in the upper micron range as well as combinations of micro- and nanostructures. UV-NIL offers additional opportunities for biotechnology devices where features with ultra- high precision are needed. Examples include optical-based biosensors that often rely on noble metal nanostructures that influence properties of coupled light upon the binding of molecules onto the nanostructures.Regardless of what the sensing principle is based on (e.g. localized surface plasmon resonance or photonic band gaps), small changes in shape and size can significantly alter the properties of the sensing element.

In order to produce nanostructures made of metals, either additive or subtractive processes can be used. The former involves the deposition of a metal layer onto the patterned resist followed by a lift-off process, whereas the latter involves the transfer of the pattern into an underlying metal layer by etching processes. In both cases, the small residual layer must first be removed. Having a uniform residual layer is of high importance, especially for subse- quent etching processes, and can be easily achieved with current equipment over large areas. Imprinted UV-NIL resists can also be used directly as functional layers. After many years of continuous resist development, a broad portfolio of optimized resist materials is available for various bio-applications. Another interesting aspect, especially for microfluidic devices, is the potential of nanostructures to influence surface properties. For example, nanostructures can change the surface behavior from hydrophilic to hydrophobic, which can be used to locally influence the fluid flow.

While UV-NIL is ideally suited for fabricating very small features, it is not well suited for features larger than several tens of micrometers. In cases where both highly-accurate nanostructures and large microfluidic channels are needed, hot-embossing can be used to imprint the channels on a separate substrate. The two substrates can subsequently be bonded together to produce the final device.

A third NIL option is μ-CP, where a pre-inked stamp is used to transfer materials such as biomolecules onto a substrate in a distinct pattern (FIGURE 4). Local modification of surface chemistry can, for example, be used to guide the growth of neurons on a chip. On the other hand, it can be used for the precise placement of capture molecules in biosensor fabrication. This technique is applicable on all common surfaces, such as silicon, glass or polymers with micro- and nanometer resolution and offers new possibilities for functionalization of biotechnology devices.

FIGURE 4: Bio-functionalized, micro-patterned array
created by micro-contact printing for the detection of protein- protein interactions in live cells. a) Antibody-patterns induce the recruitment of two interacting proteins to micro-patterns, which is detected by fluorescence microscopy. b) Missing interaction of the two candidate proteins leads to homogenous distribution on the functionalized surface. c) Schematic drawing of micro-contact printing process flow. [Images adapted from Schwarzenbacher et al., 2008, Nature Methods; Weghuber et al., 2010, Methods in Enzymology].

FIGURE 4: Bio-functionalized, micro-patterned array
created by micro-contact printing for the detection of protein- protein interactions in live cells. a) Antibody-patterns induce the recruitment of two interacting proteins to micro-patterns, which is detected by fluorescence microscopy. b) Missing interaction of the two candidate proteins leads to homogenous distribution on the functionalized surface. c) Schematic drawing of micro-contact printing process flow. [Images adapted from Schwarzenbacher et al., 2008, Nature Methods; Weghuber et al., 2010, Methods in Enzymology].

Although most current microfluidic devices do not follow the same degree of miniaturization in terms of chip-size compared to the microelectronics industry, large-scale parallel processing has a significant advantage in terms of costs and flexibility (FIGURE 5). Alternative fabrication techniques for microfluidic chips, such as injection molding, are principally serial processes and have limita- tions in up-scaling.Using nanoimprinting,30chipsofthe size of a microscopy slide (25 x 75 mm) can easily fit on a single 300-mm substrate. This format can be considered a good reference for an average- sized microfluidic chip. In terms of throughput, wafer-based batch processing is able to reach similar or better cycle times per device compared to alternative solutions, such as injection molding. UV-NIL has even been introduced on GEN2 substrates (370 x 470 mm). In addition, roll- to-roll processing can reach even higher throughput levels but is restricted to the structuring of flexible foils.

FIGURE 5: Large-area parallel processing offers significant advantages in terms of cost and flexibility. Additional processes, such as electrode fabrication or spotting of reagents, can also be efficiently integrated.

FIGURE 5: Large-area parallel processing offers significant advantages in terms of cost and flexibility. Additional processes, such as electrode fabrication or spotting of reagents, can also be efficiently integrated.

Wafer bonding

NIL has an additional advantage in terms of post- processing. Electrode fabrication, surface treatments or spotting of bio-reagents can be efficiently integrated in a large-area batch. The same is true for sealing and encapsulation, an essential process step for all biotechnology devices. It is usually mandatory to close micro-fluidic channels, to fabricate a hermetic sealing for protection against environmental influences or even to provide packaging that is compatible for implantation into human bodies. In addition, interconnections to the outer world have to be incorporated, such as holes or fluidic connectors. Electronic connections or assembling the device together with an integrated microelectronic chip is also often necessary. Thus, bonding of different device layers, capping layers or interconnection layers is a key process that can be implemented together with NIL in a cost-effective large-area batch process. NIL has an additional advantage of providing a high surface quality that can significantly improve subsequent bonding of polymer devices. Surface roughness, total-thickness variation as well as warpage are usually lower than in devices fabricated by injection molding. In the following section, several well-suited bonding processes for sealing biotechnology devices are discussed (FIGURE 6).

FIGURE 6: Typical bonding options for biotechnology devices that are well suited in combination with NIL processes.

FIGURE 6: Typical bonding options for biotechnology devices that are well suited in combination with NIL processes.

A common requirement in biotechnology applications is optical transparency, at least from one side, since most devices rely on optical readouts. Glass is therefore often used as a capping layer for highly complex devices made of silicon. In such cases, anodic bonding can provide a high-quality hermetic seal, where bonding is achieved by high voltage and heat causing inter-diffusion of ions. Another process for joining glass or polymer devices is thermal bonding using high temperatures and pressures. Special attention has to be paid when using this technique for bonding polymer and, in particular, polymer micro-fluidic devices. Thermal bonding is performed by heating the substrate near or above the glass transition temper- ature, which softens the material. The additional pressure generates sufficient flow of polymer at the interface to achieve intimate contact and inter-diffusion of polymer chains. Pressure is removed after the substrate is cooled down to a specific value below the glass transition temperature. Un-optimized temperature and pressure can easily lead to deformation of microstructures. Plasma as well as UV and ozone treatment can be used to activate the polymer surface, which allows bonding at reduced temperatures and reduces the risk of deformation. Anodic and thermal bonding are interlayer-free processes and therefore do not introduce any additional material to the device.

Adhesive bonding is another process that found widespread use in sealing or encapsulating bio-technology devices. Many biocompatible adhesives are available today and high bond strength can be expected from this technique. Bonding with adhesives can be used to join many different materials. Often liquid adhesives are used, which can be cured thermally or by exposure to UV light. The latter offers a significant advantage that addresses another important issue in many pharmaceu- tical or diagnostic devices where bio-molecules have to be incorporated before sealing the device. UV-curing allows bonding at room-temperature whereas higher temperatures usually lead to denaturation or complete destruction of bio-molecules.

Adhesives usually have to be selectively deposited on the substrate, which can be achieved with μ-contact printing. Similar to bio-molecule printing, an adhesive can be trans- ferred onto the substrate according to the pattern of the stamp. In contrast, however, an adhesive can be spin coated onto a transfer plate, which is then brought into contact with the substrate. By releasing the transfer plate, the adhesive will remain on the heightened structures. This production process is an elegant solution for micro-fluidic devices where micro-channels stay free of adhesive without the need for alignment. With these methods the adhesive can be coated as a thin layer (typically on the order of several microns) with very good uniformity over large areas. Commercially available adhesive tapes offer another solution, which can be easily laminated onto the microfluidic chips either in the form of double-side-adhesive tapes or pressure-sensitive-tapes. By using this process, the tape covers the top of microfluidic channels and can alter chemical or physical parameters of the channels, which can then influence the fluidic behavior or biological function of the device. Due to the availability of a variety of different tapes, however, such influences can be addressed and eliminated in many applications.

Summary

Micro- and nanotechnology combined with biotechnology has the potential to revolutionize many areas of healthcare, agriculture and industrial manufacturing. The market for miniaturized bio-devices is rapidly growing with technologies becoming increasingly complex. For successful translation of these technologies into new products, the availability of fabrication tools is key. Today’s NIL equipment offers a well suited solution, where complexity in design does not necessarily add manufacturing cost. Together with sealing and bonding processes that are well aligned with these structuring techniques, limitations of current fabrication methods can be overcome to enable the production for next-generation biotechnology devices.

Further reading

T. Glinsner and G. Kreindl, “Nanoimprint Lithography,” in Lithography, M. Wang, Ed. InTech, 2010.
T. Glinsner, T. Veres, G. Kreindl, E. Roy, K. Morton, T. Wieser,
C. Thanner, D. Treiblmayr, R. Miller, and P. Lindner, “Fully automated hot embossing processes utilizing high resolution working stamps,” Microelectron. Eng., vol. 87, no. 5–8, pp. 1037–1040, May 2010.
G. Kreindl, T. Glinsner, and R. Miller, “Next-generation lithography: Making a good impression,” Nat. Photonics, vol. 4, no. 1, pp. 27–28, Jan. 2010.

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