Microfabrication and microfluidics and their application in clinical diagnostics

Background

Microfluidics is a burgeoning area of analytical chemistry that will impact many fields, including clinical diagnostics. The ability to miniaturize and expedite chemistry with smaller volumes presents the possibility for testing with expedited turnaround times at lower cost and possibly in a portable and handheld format.

Content

This chapter describes the basic concepts necessary to understand microfluidics at a fundamental level. This includes the methods and materials used for fabrication, both historically and currently, as well as aspects of microfluidic architecture necessary to carry out reactions, chemistry, labeling, and detection. The chapter highlights some of the basic developments associated with the microfluidic manipulation or analysis of diagnostically relevant analytes such as cells, nucleic acid (NA), proteins, and small molecules. It also presents some exemplary applications (e.g., circulating tumor cell capture and pathogen detection) that have paved the path for the development and adoption of microfluidics in clinical chemistry and molecular diagnostics.

The first miniaturized analytical device with some degree of functionality was reported in 1979, when the separation of a simple mixture of low-molecular-weight (MW) compounds was achieved in only a few seconds in a microdevice fabricated in silicon. The significance of this development can be appreciated only by comparison with the state of the art of separation techniques common to the laboratories of that time. The 1970s saw most chemical separations carried out by open-column, paper, and thin-layer chromatographic processes; gas chromatography (GC) was only beginning to mature into a bona fide technology for small molecule analysis. Pressure liquid chromatography began to evolve into a method that would bring faster analysis times with higher resolution, and the late 1970s brought new chemistries amenable to reverse-phase liquid chromatographic separations for improved resolution of more polar compounds, including biological analytes. Electrophoresis was the workhorse for large biomolecule separations, with Laemmli having established denaturing separations of proteins in acrylamide gels and O’Farrell adding the power of a second separation dimension in two-dimensional gel separation of proteins ; the advent of sequencing gels and the use of agarose gels for nucleic acid (NA) separations followed later that decade.

With that historic context, visualize the stark contrast of a paper that reported a GC analyzer fabricated in silicon ( Fig. 27.1 ) using the tools under development for a microelectronics industry that was still in its infancy. The work of Terry et al. provided the first evidence that microfabrication could be used to provide microminiaturized devices for analytical separations. Despite that evidence, the scientific community did not immediately recognize the significance of this achievement nor see this as a glimpse into a future that would be seeded, in no uncertain terms, by Widmer’s group at Central Analytical Research, Ciba-Geigy AG (Basel, Switzerland) approximately a decade later. Although several papers followed this initial work, it was not until the concept of a total analysis system (TAS), first proposed by Manz et al. in 1990, that the idea of miniaturization had a significant impact. The TAS concept was originally motivated by a lack of adequate sensors for detection of specific species from a complex mixture. It was hypothesized in the seminal Manz et al. paper that, by improving the sample treatment steps and thereby increasing the selectivity of the sensor, an ultrasensitive sensor would not be required if interfering chemical compounds were removed. A TAS was proposed that entailed initial sampling, transport of the sample, sample pretreatment steps, and the final detection of the analyte. Miniaturization of all of the processes required for total analysis, a micro-total analysis system (μTAS), would allow development of systems that perform these functions at the site of measurement (i.e., not in a central laboratory). Additional benefits are also derived from decreasing the size of a TAS, including decreased volumes of sample and reagent and the potential for drastically reduced analysis times.

Photolithography and glass etching techniques

In standard photolithography practices, glass wafers are obtained with photoresist and metal layers already deposited on the wafer ( Fig. 27.2 ). These films function to allow for channel design and for glass protection during the chemical etching step. A chrome metal layer is first deposited onto the glass because of its tight bonding with both glass and silicon. This layer provides extra protection from hydrofluoric (HF) acid during wet chemical etching. On top of the metal is a layer of photoresist that allows for the channel patterning. The traditional mode of application is to spin coat the photoresist on top of the metal layer, although there are alternatives to this approach, such as the constant-volume injection method. As a result of the microscale precision attainable by photolithography, the application of the photoresist is performed under cleanroom conditions using specialized instrumentation.

The transfer of the channel pattern to the photoresist involves using a photomask containing the design. The photomask can either be a film mask or metal mask. For either mask, the microfluidic design can be created using standard design software (e.g., Adobe Illustrator, Adobe Systems, San Jose, CA; AutoCAD, Autodesk, San Rafael, CA). A relatively inexpensive film mask can be printed by a specialized high-resolution printer, allowing for microfluidic channels 40 μm or greater in diameter. Alternatively, metal masks can be used for higher-resolution structures (<20 μm). Then the photomask is placed on top of the photoresist or metalized wafer and exposed to a wavelength of ultraviolet (UV) light that destroys the exposed photoresist as determined by the design of the photomask. With the mask removed (and retained for further use), the wafer is then placed in a developer solution that removes the photoresist where the channels will exist. The exposed chrome is then removed, exposing the glass only in the places where channels are to be etched.

The glass channels are obtained by wet chemical etching with HF acid. Extreme caution when using HF acid is warranted. The etch rate in glass depends on the concentration of HF acid, with faster rates correlating to higher HF acid concentrations. Etch rates of 10 μm/min in borosilicate glass can be achieved with a 49% HF acid etch solution. The use of wet chemical etching results in an isotropically etched channel in which the glass is etched equally in all directions. This type of etching results in a channel architecture that is semihemispheric in shape (see Fig. 27.2 ). After the channels have been etched to the desired width and depth, access holes are drilled in a capping layer, and both glass wafers are thoroughly cleaned, aligned, and brought into intimate contact for thermal bonding. Although grounded in the scientific method, there is an “art” associated with the bonding process at temperatures between 620 and 690 °C for 6 to 8 hours. The use of a programmable furnace is critical to step ramping the temperature (up and down), and this must often be determined empirically for each system. Multiple bonding cycles may be required to get an effectively bonded microdevice, representing one significant drawback to thermal bonding. Other approaches have been developed, such as room temperature bonding techniques, UV-curable adhesives, ^, and silicate solutions.

Alternatives to traditional photolithography and wet chemical etching

If not purchased in ready-to-etch form, the application of a photoresist requires a spin coater and cleanroom conditions, which can add expense. Alternatives to photoresist have been developed, such as patterned PDMS affixed to a glass wafer that is etched by HF acid flowing through PDMS channels. Enzyme nanolithography can also achieve nanometer-sized depressions in a protein surface. With this method, scanning probe microscopy controls a nanopipette, which delivered proteases to a dried bovine serum albumin (BSA) film. ^, Although wet chemical etching does not require expensive instrumentation, the desire for high aspect ratio structures or for more control over the dimensions of the channel creates a need for other types of etching techniques. For example, high aspect ratio structures were obtained in glass and fused silica wafers by using powder blasting, fast atom beam etching, and deep reactive ion etching (DRIE). All of these methods involve bombarding the wafer surface with particles; however, whereas DRIE uses chemically reactive ions, typically fluoride, to collide and remove surface atoms, fast atom beam etching uses neutral particles.

Polydimethylsiloxane

PDMA is one of the most popular polymeric materials used as a microdevice substrate. Fabrication is simple, involving the combination of elastomeric base and curing agent. PDMS has methyl groups on the surface, imparting a hydrophobic nature, and is flexible and optically transparent. The flexibility of PDMS is useful for valving, and it can be both reversibly and irreversibly sealed to a variety of surfaces. A number of different PDMS fabrication methods have been established (see Sun and Kwok for a review), with replica molding and rapid prototyping perhaps the most popular. Rapid prototyping involves the use of a master, which once made allows for fast and easy replication of a particular design. The master is created by using a negative photoresist, such as SU-8, spun onto a silicon or glass wafer. The microfluidic channel design is imparted onto the photoresist using photolithography, and the wafer is subsequently developed. The negative photoresist retains the structures exposed to light, and the masked areas are removed by a developer solution. This leaves elevated structures where channels are desired in the final PDMS device. The height of the elevation over the glass dictates the depth of the PDMS channels. Although photolithography is needed for the fabrication of the PDMS master, after it has been created, multiple devices can be easily made.

PDMA as a solid substrate is produced when an elastomeric base and curing agent are mixed in the appropriate ratios. The mixture is poured over the master and allowed to cure; this can be accomplished overnight at room temperature or expedited using a heating process. After curing, the PDMS can then be peeled from the master and bonded with the substrate of choice. The bonding method will depend on the substrate used as the base or capping layers; PDMS-PDMS devices can be either reversibly or irreversibly bonded via a number of different methods, one being the activation of the surface by plasma oxidation. ^, Another method alters the elastomer base to curing agent ratios, using 20:1 for the fluidic layer and 5:1 for the cover plate. The device is assembled and placed in an oven, at which point the two layers “fuse” because of the distribution of the excess reagents between the two layers.

Poly(methylmethacrylate)

PMMA has become a popular substrate for microfluidic devices. PMMA is considered to be a thermoset polymer, which softens into a viscous liquid when heated above its glass transition temperature. Multiple methods of fabrication are available for PMMA, and the channel surface is easily modified to adapt to a particular application (see reviews of polymer fabrication techniques). ^, Hot embossing ^, consists of heating the polymer above the glass transition temperature and stamping the substrate with the desired microfluidic pattern. To make PMMA devices using this technique, a metal or silicon stamp is brought into contact with the PMMA, and constant force and elevated temperatures partially melt the substrate. The embossing process can be completed in less than 10 minutes, and after the apparatus has cooled, the stamp and substrate can be carefully separated. This same process can also be done at room temperature using PMMA as a substrate, yielding channel dimensions that vary by less than 2%. Injection molding, another method that can be used for fabrication of PMMA devices, also incorporates the use of a metal or a silicon master. This involves heating the PMMA pellets to temperatures that result in the formation of a viscous liquid. The PMMA is then pushed into the mold by applying pressure and allowed to cool, and it is removed by “lift-off” from the mold. Both of these methods result in devices with micron-sized channels and can be used for mass production of devices with the same microfluidic architecture. For applications that require submicron features, x-ray lithography is capable of making these intricate structures. PMMA poststructures can function as a solid phase for DNA chromatography ( Fig. 27.3 ). Solid phases for DNA extraction traditionally are created with silica beads, sol-gels, or silica bead–sol-gel hybrids. DNA is loaded in a solution that forces DNA binding to the solid phase. It is washed, and then the purified DNA is eluted off the solid phase and captured. Although traditional forms of extraction involve packing the solid phase into a capillary or tube that solution can pass through, phases can also be microfabricated during the microchip fabrication process. This is done using either a direct write process or the use of a mask, typically fabricated in a thick gold layer that functions to absorb x-rays. Where the x-rays are incident upon the PMMA, the polymer is degraded, and it can be removed with a developer solution to solubilize the degraded products. The length of exposure, along with the energy used, dictates the depth of the channels. Laser ablation is also used to make submicron features in PMMA devices. This involves bombarding the surface with a pulsed UV source to ablate (vaporize) the polymer structure. With these last two techniques, high aspect ratio features can be obtained; however, neither of these methods is ideal for mass production.

Executing an electrophoretic separation

When performing chip-based separations, the separation channel in a cross-t design provides the counterpart of the capillary in CE but with a substantially truncated separation length ( Fig. 27.4 ). Many of the parameters governing electrophoretic separation in capillaries apply to chip-based separations. For example, similar to capillaries, one of the benefits of glass-based devices is their inherent ability to dissipate the Joule heat (because glass is an effective conductor of heat) generated by the high electric fields needed to facilitate rapid separation. However, the same type of Ohms law plot (applied voltage vs. current) used to test buffer compatibility with high electric field applications should be carried out here as well. If performing separations requiring electro-osmotic flow, the rigorous cleaning and hydration methods suggested for fused silica capillaries is required to ensure proper surface regeneration. The entire cross-t channel architecture of the device is filled, including the reservoirs, to ensure good electrical contact between the platinum electrodes and the solution. The holes drilled into the microfluidic device to function as reservoirs (i.e., buffer and sample microvials) may not be large enough to hold a sufficient volume of buffer to avoid buffer depletion. The user must remain cognizant of this and, even with expanded reservoirs generated by commercial “nanoports” or homemade trimmed pipette tips, the replenishment of buffer may be needed.

For sample injection, separation, and detection in a chip of simple design (see Fig. 27.4 ), sample is placed in the reservoir, and a voltage is applied between sample inlet (SI) to sample outlet (SO) to inject the sample into the separation channel in a manner consistent with the injection mode chosen. Separation is initiated by application of voltage to buffer outlet (BO) and buffer inlet (BI), where electrophoresis of the sample analytes down the separation channel ensues. A point along the separation channel, most commonly at a location close to BO, is used for detection. The most frequent detection mode is optical, with laser-induced fluorescence (LIF) popular because of its sensitivity. The detector system (e.g., a laser and photomultiplier tube [PMT]) is positioned at a point in the separation channel commensurate with providing the necessary effective separation length (L _eff ) for the separation; the sample analytes passing that detection point are excited and the resultant fluorescence collected. Similar electric fields ( E ; volts/cm) are applied in both CE and ME; however, reduction of the L _eff is more easily achieved in microchips. The reservoirs and electrodes required to apply voltage to capillaries limits the minimal L _eff to tens of centimeters; microchips, in contrast, can easily complete separations in a few centimeters. Interestingly, the U-shaped channel in microchips has little effect on the resolution of separations under high electric fields. Similarly, the plug profile typically associated with capillaries and known to play a role in high-efficiency separations is not adversely affected by hemisphere-shaped channels.