Medical Device Link.

Originally Published IVD Technology March 2003

Processing Technologies

Early experimental results support the possibility of eventual mass production for clinical diagnostic applications.

M. Goretty Alonso-Amigo and Tom Adams

Figure 1. Optical image of a quarter section of the plastic rotating chip developed in the reported study.  (Click to enlarge)

Glass rotating chips have been used for several years in advanced microfluidics applications, particularly where high throughput is desirable. The microfluidic chip concept has been cost-effective in terms of reducing reagent consumption and shortening the time required for analysis. However, as rotating chips come to play a greater role in routine clinical diagnostic applications via lab-on-a-chip technology, very large production volumes will be needed. The additional economies demanded by that development will necessitate replacing glass construction with plastic.
A study was undertaken to demonstrate that rotating chips could be microfabricated in plastic materials using conventional injection molding technology and thermoplastic resins, as have been used with other microfluidics designs.¹ The chip design used as the basis for the investigation was that of a glass rotating chip with 96 capillary electrophoretic channels that was introduced several years ago.²

Rotating-Chip Technology

The rotating-chip concept was designed—along with a confocal fluorescence detection system—to address several limitations of planar capillary array systems. Rotating chips have evolved into 384-channel devices and are regarded as next-generation tools for genetic analysis, including gene mapping, pharmacogenomic screening, and applications in forensics and proteomics.³

All publicized multichannel rotating chips have been made of glass. Standard microfabrication procedures have been employed to etch trenches 30 to 50 µm deep on the surfaces of glass wafers measuring either 100 or 200 mm in diameter. The sample, waste, and cathode wells in these chips are located at the perimeter, while all separation channels converge on a single anode well in the center of the chip. More recently developed rotating-chip designs include all sample preparation steps and even integrated microelectrodes.^4,5

Glass rotating chips as large as 200 mm in diameter have been microfabricated successfully. As the wafer diameter increases in the rotating-chip format, sample-preparation elements can be allocated to the microfluidics structure, fewer channel turns and longer separation channels allow for such high-resolution applications as DNA sequencing, and the accommodation of more channels than can be provided by other microfluidic designs makes possible high-throughput systems. Thus, rotating-chip designs facilitate development of fully integrated and high-throughput analytical systems.

Some plastic circular microfluidics designs have been commercialized. Gyrolab, from Gyros AB (Uppsala, Sweden), is used for sample preparation in matrix-assisted laser desorption/ ionization (MALDI) analysis of protein. Tecan Systems (Durham, NC) offers the LabCD for Cytochrom P450 inhibition and serum protein binding assays. Both of these systems enhance analytical productivity through microfluidic liquid handling. Each is an injection-molded and fully integrated plastic card about the size of a compact disc.

Figure 2. Diagram of a channel in the microfluidics chip. During thermal bonding, channel walls are vulnerable to distortion, especially where channels meet. (Click to enlarge)

The 96-channel rotating-chip design used in the study reported here has a separation distance, from the injection point to the anode well, of 67 mm (see Figure 1). The microfluidics chip consists of four quadrants, each of which contains 24 channels and 48 small wells 1.2 mm in diameter. The wells will contain biologic samples, such as blood, and reagents, which are moved along the channels by means of an electrical field generated by electrodes embedded in the wells. With a chip of this design, the sample introduced for analysis must be ready for the separation conditions.

The chip itself is a separation device, one designed to cut costs dramatically by virtue of the tiny quantities of reagents used. It also reduces analysis time from the current 1-hour standard to 20 minutes or less. This new biotechnology tool can be used, for example, in genotyping, where reagents (enzymes in this case) break the DNA into smaller chains that are separated in the channels to produce the particular genetic pattern of the DNA sample. Development of the microfluidics chip is aimed toward high-volume, low-unit-cost production.

Application in Diagnostic Systems

Microfluidics, microarrays, and biosensors are three distinct technologies that converge in the system concepts called lab-on-a-chip (LOC) and micro total analysis systems (µTAS).6–8 LOC and µTAS devices carry the promise of replacing the complex analytical systems now found in research and clinical laboratories and in doctors’ offices. Future LOC devices will contain miniaturized, microfabricated functional elements capable of performing all the steps needed to go from a physiological sample (blood, urine, tissue) to analytical information. These devices have immediate application in genomics and proteomics discovery, in the medical diagnosis and prevention of diseases, and in making therapeutic treatments effective.^9–11

Most prospective diagnostic LOC applications are complex. It is expected that microfluidics, microarrays, and biosensors will be integrated in many diagnostic products. For example, in diagnostic systems for point-of-care or home testing, a clinical sample might first be measured with electrolyte-specific biosensors and then undergo, via microfluidics, preparation steps—separation of components, preconcentration, amplification, and prelabeling—necessary prior to bioanalytical testing on a DNA or protein microarray.

LOC products with little or no integration are already commercially available for research use in genomics and proteomics. As both understanding of the technology and the capacity to accommodate the complexity of integrated analytical functional elements develop, LOC systems will come to correspond more closely with total analysis systems as they are conceived, with reduced dimensions and increased analytical capacity. To get to this level of development will also require development of integration and assembly processes that will allow cost-effective manufacturing of LOC devices in the very large volumes that will be needed for clinical diagnostic applications.

Plastic versus Glass

Since the LOC field was born, plastic microfabrication has been proposed as the approach to take in minimizing the cost of manufacturing disposable LOC chips. Cost reduction can be expected not only from the lower cost of materials but also, more significantly, from the more cost-effective serial mode of microfabrication possible with plastic. When thermoplastic materials are injection molded, or shaped by another compatible molding technique, from a mold prepared via standard microfabrication techniques, micron-size features can be replicated with high quality.

The most typical production approach begins with a microfluidics design of capillary channels and micron-scale structures being microfabricated into a glass master. This glass master, by means of an electroforming process, is converted to a negative metal master that accurately duplicates all of its features. This metal master is adapted to serve as the molding tool in an injection molding system.

A viscous-plastic molding resin is injected into the mold, conforms to its fine structures, and then solidifies into accurately detailed plastic microparts. This production cycle is completed in less than a minute. Potential manufacturing output is 5000–10,000 products per day when the molding operation is fully automated. With batch production in glass microfabrication, by contrast, production runs between 75 and 500 etched glass plates per day. Other factors involved in determining final chip production, including microfabrication yields, the length of chip bonding and dicing cycles, the number of chips per molded plate or glass wafer, and the duration and production yield of other integration steps, are not included in this comparison of production volumes.

The cost difference between glass and plastic microfabrication depends on volume. Plastic has the advantage of lower variable cost per microfabricated part in production. However, the fixed cost of tooling for plastic microfabrication is significantly greater than that for batch production of glass plates. Thus, glass microfabrication is more cost-effective for low-volume chip production. But this economy is nonscalable; when high volumes are needed, plastic microfabrication has a large edge.

Chip production for future LOC applications will include all necessary integration steps. These may entail electrode microfabrication steps for electrophoretic chips; steps involved in the addition and storage of reagents in the chips; plus other bonding, sealing, assembling, and packaging steps. Electrode microfabrication may or may not involve metallization. Stamp-printing of conductive materials for microfabricated contacts is known to work for some microfluidics applications.

Figure 3. An acoustic image shows distortion in channel walls at an early stage in developing the thermal bonding process. (Click to enlarge)

Metal-electrode fabrication processes might be required for certain applications, however. The use of glass favors the more stringent conditions of surface metallization, but plastic-surface metal sputtering processes are routine in certain industries, and it is foreseeable that these processes could be adapted to micropatterning. Thus, no cost difference between glass and plastic chip production is attributable to this step in chip integration.

So far, the availability of microfabrication equipment and the need for only small volumes of chips for product development has favored the commercialization of glass rather than plastic chips. High up-front product development costs in time and tooling that have hindered progress in generating LOC devices based on plastic microfabricated components are another factor in the relatively late introduction of plastic microfluidics technology.

The Plastic-Chip Manufacturing Process

The preparation of plastic rotating chips for the study under discussion involved standard injection molding using standard polymethylmethacrylate thermoplastic resin (PMMA acrylic). The microfabrication process began with the injection molding of plastic wafers with micron-size trenches that would later define the capillary channels; these were the channel plates. Next came the injection molding of flat wafers from PMMA. These featureless pieces served as cover plates. The third step was the drilling of holes at the ends of the channels in the microfluidics design, performed via standard CNC machining. Finally, the capillary channels of the chips were given finished form through a bonding process in which cover plates were thermally fused to channel plates (see Figure 2).

Figure 4. An acoustic image revealing that heat has collapsed the walls of adjacent channels (A) and allowed material to flow into the converging anode well (B).  (Click to enlarge)

The unprecedented achievement of the project was the replication of micron-size features in 300-mm plastic wafers that exhibited flatness of better than 100 µm edge to edge along the wafer diameters, and 92% light transmittance throughout the entire 700 cm2 of available surface in the molded wafer. The cover plates produced by injection molding shared these properties.

Bonding involved use of a large platen press and followed general guidelines for the thermofusion of thermoplastic parts. In this process, two plastic surfaces join with application of the minimum necessary pressure and heat. Under optimal bonding conditions, the surface layers of the polymers flow enough to fuse with each other. These conditions are attained by heating the plates, very uniformly, to a temperature above the glass transition temperature of most PMMA resins (105°C).

The glass transition temperatures of polymer resins are specified by their manufacturers. They also can be measured using thermal-analysis tools. The exact optimal bonding conditions of pressure and temperature for a given thermoplastic depend on the thermal capacity of the press, the thickness of the two elements being bonded, and the design of the microstructures being formed. Bonding parameters need to be optimized for any new design. Experimental results have suggested that a temperature fluctuation of 1°C can make the process fail.
Successful microfabrication of microfludics chips depends on being able to reproducibly form the channel structure through precision bonding. In forming the channels, total surface bonding of the zone near the channel walls is ideal. The fused interface of the plates is, at the molecular level, an entanglement of polymer chains from both surfaces. Given that the surfaces to be bonded are brought into contact appropriately, very low pressures of 1000–2000 psi suffice to effect the bond once the material is in the viscous state. Pressures greater than that induce flow at weak points in the channel walls or at the intersections of channels and wells, which can reduce the channel cross section or even block the channel completely, preventing microfluidic flow.

Quality Evaluation by Acoustic Microimaging

The geometric integrity of the channels and wells is vital for the predictable flow of fluids. Although the PMMA acrylic in the two-piece bonded wafer is largely transparent to light, distortions in critical elements such as the walls of the channels cannot be detected optically. But, like most nonporous production materials such as polymers, metals, and ceramics, the acrylic is also transparent to ultrasound. It was therefore possible to characterize the channels and wells by means of acoustic microimaging. Very-high-frequency ultrasound is sensitive to changes in modulus and density, and is useful in characterizing the elastic properties of materials. A D-9000 C-mode scanning acoustic microscope from Sonoscan Inc. (Elk Grove Village, IL) was used to perform acoustic microimaging of sample plastic chips. It was operated with a specialized ultrasonic transducer for die-level imaging.

Principles of the Technique. 

Figure 5. Improved thermal control during bonding prevented the problem of collapsing channel walls near the anode well illustrated in Figure 4. This acoustic image was made after bonding by means of the later process.  (Click to enlarge)

Acoustic microimaging uses very-high-frequency (5 to 100 MHz) or ultra-high-frequency (100 MHz-plus) ultrasound. An ultrasonic transducer performs two functions: it pulses ultrasound waves into the sample, and collects the return echo signals from within the body of the sample. The transducer performs each of these functions several thousand times per second while raster-scanning the sample.

Echo signals of various intensities are returned only from material interfaces. Signals traveling through a homogeneous material such as a single layer of acrylic in the wafer return no echoes. At a material interface, the acoustic velocity of each material and the density of each determine the degree of signal reflection.

Acoustic microimaging is used in electronics applications where an interface may be formed from a polymer and a metal, or from a ceramic and a polymer. In the microfluidic chip, the two interfaced materials are identical acrylics. If these identical materials are well bonded, that is, if bonding has achieved the right level of polymer chain entanglement, then the echo signal at their interface will be very slight or entirely absent.
The cover plate interfaces not only with the acrylic material of the channel plate, but also with empty spaces defined by the channels and wells. In the latter regions, the interfacing materials are acrylic and air. The amount of acoustic reflection is still governed by the density of the materials and their acoustic velocity. However, air density is extremely low, and the speed of ultrasound through atmospheric gases—because the molecules are so far apart—is typically zero eliminating the wave or very close to zero allowing for minimal transmission of ultrasound. The result of these values is that the reflection of ultrasound from an air gap is typically very high; virtually 100% of the energy at the interface goes into the echo signal. This is true even if the gap is measured in nanometers.

Figure 6. Contamination within a well is revealed acoustically as small areas of echo signals.  (Click to enlarge)

For the acrylic chip as a whole, then, acoustic microimaging will show an empty field (no echo signals) over well-bonded areas and a strong contrast over gaps such as channels and wells, or even trapped dust particles or air bubbles, which return very strong signals. Variations and distortions in channel walls, or anomalies in the plate bonding, will also be visible. Acoustic microimaging results are unrelated to the optical properties of materials being characterized. In the case at hand, they would be the same if the acrylic plates were optically opaque.

Ultrasound waves travel through most production materials at very high speeds—on the order of thousands of meters per second. Since the distance from the ultrasonic transducer to the interior of the sample is typically a matter of millimeters, a round trip usually takes a few microseconds. This is why the transducer can raster-scan the sample at high speed and still perform both pulsing and echo-capture functions.
During the brief interval of the pulse-echo function, echo signals from different depths within the sample arrive back at the transducer at slightly different times. Users of acoustic microimaging often take advantage of this time spread to electronically gate the echo signals on a particular depth of interest, rather than using signals from all depths. In addition, the transducer is focused on the same depth of interest. Gating and focusing were used in the present study to make acoustic images at the depth of the interface between the two acrylic plates. Acoustic images may be displayed in monochrome or in a variety of artificially colored maps.

Acoustic images gated at this interface were acquired at an early stage of plastic rotating-chip development when the relationship between thermal control and channel wall integrity was not well understood (see Figure 3). The channels form injectors having the shape of a double T. At these points the walls of the channels tend to be weaker and more easily deformed. Images revealed wall collapses, as in the figure. The straight white lines expected in an acoustic image of the channels were replaced by irregular areas whose color indicated the reflection of material that had collapsed into the channel. Also visible were other deformations of the channels at some distance from the double-T injectors and channel T-joints. Small anomalies away from the channels altogether may have been voids or dust particles trapped between the two acrylic plates during assembly.

Acoustic microimaging also showed that channels converging near the anode well were susceptible to wall distortion (see Figure 4). The acoustic image in the figure represents an early developmental stage; the thin walls have collapsed, blocking the channels and enabling material to migrate into the well. Only the leftmost channel remained open following bonding, a fact determined by using liquid-flow testing methods. Acoustic imaging of this anomaly led to tighter thermal control and better bonding results (see Figure 5).

Not every problem that arose during the effort to develop successful plastic-chip manufacturing processes involved the channels. It was found that particles or impurities traceable to the lack of cleanroom environmental control during the bonding process could infiltrate the wells (see Figure 6).

Basic optical and flow testing of the microchannels of chips manufactured flawlessly by means of finally optimized production processes were performed. This testing involved a confocal microscope and use of a circular electrode array to apply electric fields.2 The results were encouraging for fluorescence detection and DNA separation analysis. Matrix formulation and separation conditions were not directly transferable from glass to plastic microstructures as expected, however. Thus, further analytical separation experiments must be conducted to assess the full performance capabilities for electrophoretic separation and the continuing development of chip integration steps for high-volume LOC applications.

Conclusion

Significant economies can be realized with the commercial manufacture of a microfluidics chip using acrylic materials and conventional injection molding techniques instead of the conventional glass manufacturing process. Control of production processes must be extremely precise, however, in order to avoid distortion of critical chip features such as channels and wells. The use of acoustic microimaging allows internal features to be clearly visualized, including any anomalies in the structure of channels and wells. Precise inspection of chip structures by means of that technology accelerated the development of the chip. Acoustic microimaging is also useful for providing process control feedback during manufacturing.

References

1. TD Boone et al., “Plastic Advances Microfluidic Devices,” Analytical Chemistry 74, 3 (2002): 78A–86A.
2. Y Shi et al., “Radial Capillary Array Electrophoresis Microplate and Scanner for High-Performance Nucleic Acid Analysis,” Analytical Chemistry 71 (1999): 5354–5361.

3. CA Emrich et al., “Microfabricated 384-Lane Capillary Array Electrophoresis Bioanalyzer for Ultrahigh-Throughput Genetic Analysis,” Analytical Chemistry 74 (2002): 5076–5083.

4. ET Lagally, I Medintz, and RA Mathies, “Single-Molecule DNA Amplification and Analysis in an Integrated Microfluidic Device,” Analytical Chemistry 73 (2001): 565–570.

5. BM Paegel, SHI Yeung, and RA Mathies, “Microchip Bioprocessor for Integrated Nanovolume Sample Purification and DNA Sequencing,” Analytical Chemistry 74 (2002): 5092–5098.

6. DR Reyes et al., “Micro Total Analysis Systems. 1. Introduction, Theory, and Technology,” Analytical Chemistry 74 (2002): 2623–2636.

7. PA Auroux et al., “Micro Total Analysis Systems. 2. Analytical Standard Operations and Applications,” Analytical Chemistry 74 (2002): 2637–2652.

8. LJ Kricka and P Fortina, “Microchips: An All-Language Literature Survey Including Books and Patents,” Clinical Chemistry 48 (2002): 1620–1622.

9. T Vo-Dinh and B Cullum, “Biosensors and Biochips: Advances in Biological and Medical Diagnostics,” Fresenius Journal of Analytical Chemistry 366 (2000): 540–551.

10. KK Jain, “The Role of Protein-Chip Technology in Molecular Diagnostics,” IVD Technology 8, no. 6 (2002): 49–56.

11. PJ Johnson and Lo, “Plasma Nucleic Acids in the Diagnosis and Managalytical Chemistry 366 (2000): 540–551.

10. KK Jain, “The Role of Protein-Chip Technology in Molecular Diagnostics,” IVD Technology 8, no. 6 (2002): 49–56.

11. PJ Johnson and Lo, “Plasma Nucleic Acids in the Diagnosis and Management of Malignant Disease,” Clinical Chemistry 48 (2002): 1186–1193.

M. Goretty Alonso-Amigo, PhD, is the founder and president of Arlanzon Technologies Inc. (Santa Clara, CA). She can be contacted at galonso@arlanzon-tech.com. Tom Adams is a freelance writer in Lawrenceville, NJ. He can be contacted at teadams@earthlink.net.   

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