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Originally Published July 2000

Semiconductor packaging technologies advance DNA analysis systems

A microchip system combines microelectronic technologies and molecular biology to execute rapid DNA hybridization analyses.

With an eye toward the commercial potential of broad markets in medical diagnostics, biomedical research, genomics, genetic testing, and drug discovery, instrumentation companies are pushing hard to develop testing technologies that will take advantage of current advances in biotechnology. A number of companies are developing automated instruments based on the use of DNA-chip technologies, each hoping that its approach will develop into a winning product.

NanoChips subassembly with NanoChips in the background.

One such company is Nanogen Inc. (San Diego), a biotech company whose researchers have developed a DNA-chip system that integrates advanced microelectronics and molecular biology into a platform technology with broad commercial applications. The company plans to begin selling its NanoChip system this year to scientists and genomics laboratories that are conducting research into single nucleotide polymorphism (SNP) scoring. According to the company, beta testing of the system indicates that it can provide accuracy equal to or better than DNA sequencing and other methods currently used for SNP detection.

The Nanogen system uses electronically accelerated hybridization under very low salt conditions, which helps to avoid most of the problems with DNA conformation and secondary structures encountered by other methods. Moreover, DNA can be arrayed and analyzed on the system's cartridges in a user-selectable format in a single day. And the system offers walk-away automated analysis.

The initial marketing focus for the system will be in the area of DNA sample analysis. Future versions of the system could provide speedy tests for the presence of viral or bacterial pathogens in tissues, blood, or other body fluids. The system could also be used in environmental, industrial, or agricultural applications, or to detect biological contaminants in food and water.

How the System Works

The NanoChip system consists of two instruments, a loader and a reader (see Figure 1). The loader enables the user to place target DNA probes, such as those from cancer cells or HIV, into the microarray. The reader uses dual lasers to excite fluorescent markers, which are detected and recorded by onboard systems.

Figure 1. Nanogen instrumentation.

Currently, chip-based biological assays that use DNA commonly require significant preparation of the sample materials, which can include blood, urine, plants, or bacteria. Processing begins with the extraction of DNA from the cells in the sample. The DNA of interest is then amplified using known processes such as polymerase chain reaction (PCR) or strand-displacement amplification (SDA). Next, the amplified DNA material is denatured (i.e., the double helix strands are separated) and placed in an appropriate buffer solution. At this point, the prepared sample can be transferred to the chip for DNA analysis.

The NanoChip system takes advantage of the fact that most biological molecules are either positively or negatively charged. These electrical charges can be manipulated through the use of microelectronics, making it possible for biological molecules to be moved in a controlled manner and concentrated at designated assay sites on the surface of the semiconductor microchip. The assay sites may be arranged in a variety of configurations, or arrays, on the microchip.

When a NanoChip is used to analyze the DNA present in a sample, small sequences of DNA capture probes are electronically moved to specific sites on the microchip. The test sample can then be analyzed for the presence of target DNA molecules by determining which of the DNA capture probes on the array hybridize (bind) with complementary DNA in the sample. The use of this electronically mediated active hybridization process to move and concentrate target DNA molecules accelerates detection, which occurs in minutes rather than the hours required for passive hybridization techniques. In addition to DNA detection, future applications may include antigen-antibody, enzyme-substrate, cell-receptor, and cell-separation assays.

This system also enables users to develop custom DNA assays. Using the loader, a blank cartridge can be configured with the desired capture probes for a particular test. Up to four cartridges and a microwell plate containing the capture probes are placed in the instrument. The loader then transfers the probes to the flow-cells of the cartridges and, using electronic addressing, directs the charged molecules in the sample to specific electrode locations. The biological sample to be tested can be introduced into the cartridge flow-cell by the loader or by manual pipetting.

The reader is used to measure the results of biological assays conducted on the microchips. When electrical fields are applied to the selected electrodes, target DNA sequences in the sample are quickly hybridized to their complementary capture probes. Using its dual laser to scan the electrode array, the reader can then detect reporter probes with fluorescent labels that indicate the sites where DNA matches have occurred. A photomultiplier detects the light emitted by the fluorescent labels, measures its intensity, and passes that measurement to onboard software.

Postprocessing software is used to analyze the signals created during laser scanning, and to assist users in interpreting the data. All of these functions —multiplexed DNA hybridization, electronic control of hybridization stringency, sample detection, and computer analysis—can be automatically performed within minutes.

Key Components

Creating a cartridge that would function as described above offered significant technological challenges. At the very least, each single-use cartridge had to meet all of the following design requirements.

• Contain liquids immediately adjacent to the microchip's surface.
• Prevent nonspecific binding of biosolution components.
• Minimize part count and associated unit costs.
• Use design-for-manufacturability (DFM) assembly features.
• Incorporate an underfill to form a fluidic chamber with a controlled meniscus, leaving functional areas of the chip uncovered and clean, and maintaining acceptable chemical compatibility.

The initial prototype cartridge was constructed with a molded polycarbonate fluidics flow-cell, a 6-mm silicon microchip, a nickel- and gold-plated polyimide flexible interconnect circuit, 120 interconnect polymer bumps, and an underfill to form the flow-cell seal (see Figure 2).

Figure 2. The underside of a NanoChip subassembly showing the integrated assay chamber formed by the chip, underfill, and substrate.

Microchips. At the heart of each disposable cartridge is a proprietary semiconductor microchip. These active microchips are made of silicon, formatted as DNA arrays, designed and constructed using microlithography techniques, coated with a permeation layer to which capture probes are attached, and mounted within the ceramic-circuit microfluidic disposable cartridge (see Figure 3).

Figure 3. Close up of a tray of completed Nanogen microchip assemblies.

NanoChips constitute hybrid circuits, meaning that they have both electrical and fluidic connections to the reader instrument. In order to complete an electrical circuit, they must be electrically interconnected on an insulating substrate onto which combinations of conductors and resistors have previously been deposited. This interconnection step is crucial in the manufacture of hybrid circuits—many hybrid circuits fail because of faulty bonds. Engineers have to consider many factors when designing the manufacturing processes for interconnections.

Interconnection Bumps. In flip-chip assembly, devices with specially processed bumps on the face of the chip are bonded face down to corresponding pads on the substrate. Wire bonding, another interconnection technique, makes interconnections with very fine gold or aluminum wire. Because such wire bonds would require bonding the face of the chip against the sample aperture in the substrate, thereby interfering with the formation of an integrated assay chamber, flip-chip bonding was found to be preferable for assembling the NanoChip.

Similarly, the use of polymer bumps was proven superior to solder bumps, and became the technology of choice in this application. The polymer process is cleaner than the solder-bumped flip-chip process, which requires electrochemical cleaning to remove solder flux residues. Other reasons for the selection of the polymer bump technology include the following.

• Lower total manufacturing costs.
• Fewer processing steps (no under-bump metallization [UBM], wafer-bumping, fluxing, or postassembly cleaning steps required).
• Lower-temperature processing, permitting the use of less-expensive cartridge materials.
• No flux or vapor contamination in the chip assay area.
• Silver polymer bumps can be used over a wide range of temperatures.

The conductive polymers used to form polymer bumps are optimized for stencil printing at extremely fine pitches. And, unlike solder, they do not flow with heat. Very dense input/output patterns can be achieved with one-pass stenciling, thus enhancing chip performance, miniaturization, and manufacturing efficiency. The use of specially formulated polymers is the key for polymer flip-chip technology. The isotropic silver-filled conductive polymer selected for the NanoChip is supplied by Epoxy Technology Inc. (Billerica, MA).

The flexible interconnect for the prototype cartridge was constructed with 4.0-mil-diam silver polymer bumps that were achieved using an electroplated stencil. Lollipop flexible fingers (flexible circuits that trace substrates) measuring 4.0-mil diam were etched in copper on a 7.0-mil pitch. The flexible circuit was constructed as a stiffened multilayer incorporating four layers of 5.0-mil polyimide film. A cured, bonded bump height of less than 1.0 mil was maintained to permit proper underfill flow properties and meniscus shape.

Underfill. Another critical area of the silver polymer bump process was the compatibility of the underfill material. This material has to perform the normal multipurpose functions of an underfill, but the ultimate goal is to attain the electrical and mechanical properties necessary for long-term reliability of the assembly during varying environmental conditions. For this application, however, it was crucial that the underfill not absorb or contaminate the DNA during analysis.

Several underfill materials were tried before one was selected that met all the criteria for this application. Like the polymer used to form interconnection bumps, the epoxy underfill selected for this application is supplied by Epoxy Technology Inc. The final underfill assembly was certified using a cyclic voltametry test, which checks the cleanliness of the electrode surface on the chip.

Chip Refinements

Although the prototype 6-mm chip and polyimide flex assembly worked well, in early 1999 Nanogen introduced a 7-mm chip assembly using an alumina ceramic interconnect substrate. As noted below, adoption of this refined model improved the efficiency and yield of the flip-chip manufacturing process in several ways (see Figure 4).

Figure 4. Comparison of a 7-mm chip on a ceramic substrate with a 6-mm chip on a polyimide flex substrate.

• Increased adhesion of the chip to the substrate made possible by using 6-mil pads with a 10-mil pitch.
• Faster processing, possible because alumina and silicon substrates have a closer coefficient of thermal expansion (CTE), permitting a higher cure temperature (snap cure) of the silver polymer bumps.
• Alumina substrates stayed flat and coplanar to the chip during curing, eliminating the need for costly special fixtures.

Adoption of the refined product also made it possible to use an inexpensive thick-film substrate (using classical thick-film technology without any etching) instead of the more expensive fine-pitch polyimide flexible or etched thick-film ceramic. The simple print-and-fire process used for such thick films can easily accommodate the 6-mil lines and 4-mil spaces required for the 7-mm chip size. Furthermore, the recent development of fine-line thick-film conductors with calendered and high-strength screens is making it possible for manufacturing to achieve even 2-mil lines and spaces, and thereby extending this lower-cost technology into the mainstream of most high-throughput flip-chip applications.

By using alumina substrates together with the low-cost silver polymer bump process, Nanogen was able to meet its cost objectives for the cartridge assembly. Conversion to the 7-mm chip and associated alumina interconnect substrate also makes it possible for Nanogen to carry out direct spin-on attachment of the permeation layer.

Completion of the Cartridge

After the chip and substrate have been assembled to form a complete flip-chip component, Nanogen applies the permeation layer on the chip and assembles the substrate to the plastic body of the cartridge.

The company's proprietary permeation layer, which is critical to the proper functioning of the Nanogen system, is the interface between the surface of the microchip and the biological test environment. The permeation layer isolates biological materials from the harsh electrochemical environment near the electrode surface, and provides the chemistry necessary for the attachment of capture probes.

The capture probes or other capture molecules are electronically addressed to the desired microlocations, and chemically attached to the permeation layer. Because independent control can be applied at any test site on the microchip, different capture probes can be addressed on the same microchip, allowing multiple tests to be processed simultaneously.

Conclusion

Nanogen expects to offer its cartridges with preloaded sets of capture probes as well as in an empty form that can be customized by the end-user. Such "build-your-own-chip" applications will allow the assembly of specific probes onto a microchip to perform individualized analyses.

In the future, the company believes that applications for the NanoChip system will capitalize on the increasing availability of genetic information to transform human healthcare. Mayo Clinic researchers recently presented the results of beta testing on clinical patient samples using the NanoChip system. In each study, the system was 100% accurate, versus an error rate of 6–11% for restriction fragment length polymorphism genotyping, which is the industry standard technique. The system is also being tested by the University of Texas Southwestern Medical Center (Dallas), and by the Bode Technology Group (Springfield, VA), an internationally recognized forensic laboratory.

It is hoped that such rapid and highly accurate systems will make a contribution to both improved patient outcomes and significant reductions in healthcare costs. A $100 assay that detects a disease while it is still in a treatable or curable stage obviously has patient benefits, and could also save thousands of dollars in downstream medical costs. This is the approach being taken by the Mayo Clinic (Rochester, MN), which is planning to introduce the use of microarray-based testing into its clinical practice in the near future.

Richard H. Estes is chief operating officer at Polymer Flip Chip Corp. and vice president for technical operations at Epoxy Technology Inc. (both in Billerica, MA).

Photos By Carol Crawford Courtesy Polymer Flip Chip Corp. (Billerica, MA)