Claude Nguyen

Writer’s comment: Writing this review paper for English 104E under the direction of Dr. Sondra Reid, I realized how important it is for writers to possess true interest in their topic. A passion for the topic, no doubt, fuels the meticulous attention to detail that is required for technical writing. Searching for my topic, I wanted to learn about the latest developments in molecular biology, particularly in the technology behind most research. I decided on microchip electrophoresis because of its relation to biochemistry, my field of study. In addition, I was interested in learning about the strides being taken to improve the efficiency of current equipment. Microchip electrophoresis promises to increase productivity while minimizing material use. The significance of this technology is clear, since long operating times and expensive biological samples are key factors limiting the science that can be performed with conventional electrophoresis.
—Claude Nguyen

Instructor’s comment: Facing the review paper assignment in scientific writing (English 104E), Claude Nguyen chose to discuss a fascinating group of studies that report advances in microchip electrophoresis, an emerging field that blends molecular biology and computer engineering. Not coincidentally, this combination reflects Claude’s achievements in both fields. In applying his continuing interest in computer science (his original major at UC Davis) to problems associated with his biochemistry major, his excellent review provides an instructive look at how technology is working to improve procedures for separating and analyzing biological samples. Such are the ways a committed student of science—particularly, one who writes clearly—helps the rest of us glimpse some of the amazing developments taking place in areas of research characterized by radical change. I, for one, am grateful for the overview and expect that other readers will be, too.
—Sondra Reid, English Department



Introduction
         Microchip-based electrophoresis represents the latest effort toward cheaper and faster separation assays. Unlike traditional electrophoresis techniques such as heteroduplex analysis (HDA) and single-strand conformation polymorphism (SSCP), which typically utilize slab gels (S. Liu et al., 2000), microchip electrophoresis employs microfabricated wafers as a separation medium for molecular samples. Current slab gel techniques possess notable faults, including slow rate of analysis, high materials cost, and difficulty in finding heterozygous DNA (Tian, Brody, et al., 2000). Recent strides in separation technology like capillary electrophoresis (CE) focus on reducing costs by miniaturizing the procedure. These processes are fast, have high precision, and reduce the amount of reagent used (Y. Liu et al., 2000).
         Because problems such as capillary wall adsorption plague these techniques (Y. Liu et al., 2000), however, demand has increased for a sequencing method that provides high throughput at low cost. Microchip electrophoresis promises to eliminate some of these flaws. By utilizing a small conductive medium such as a silica wafer to conduct samples, the method consumes only minute amounts of sampling material and reagents. Microdevices also tout high throughput levels and increased automation (Tian, Brody, et al., 2000). Using short separation channels and narrow sample bands, microchip electrophoresis can sequence samples more quickly than traditional techniques (S. Liu et al., 2000). Although microchip electrophoresis possesses these advantages, its purported improvements in precision have not been proven (Schmalzing et al., 2000). Issues such as standardization in chip design and problems with resolution must also be addressed before the technology can be widely embraced.

Performance of Microchip-based Electrophoresis

Separation Rate
         Microchip electrophoresis separation times are much shorter than those of traditional techniques. In one experiment, microchip-based SSCP performed on both wild-type and normal 185delAG, 5382insC, and 6174delT alleles was completed in less than 120 seconds (Tian, Jaquins-Gerstl, et al., 2000). This is four times faster than conventional SSCP and 100 times faster than traditional gel electrophoresis. Another project by Schmalzing et al. (2000) compared microchip electrophoresis to conventional slab gel separations in detecting mutations in clinical samples. Results showed that microchip electrophoresis was fifty times faster than slab gel methods while maintaining a comparable accuracy level (± 5 base pairs). While the gel method took 64 minutes to resolve a mutation, the same mutation was detected on microchip in only 68.4 seconds.

Separation Resolution
         Although most applications of microchip electrophoresis report equal or greater sample resolution compared with traditional sequencing, actual resolution using microdevices may vary. Resolution achieved using microchip electrophoresis seems to depend on the characteristics of the solutions used. Rodriguez, Jin, and Li (2000) tested the sequencing of amino acids using varied concentrations of borate buffer. As they increased the concentration of borate, the resolution for all samples tested improved. In addition, samples with higher ionic strengths produced faster results and higher resolution (Rodriguez, Jin, & Li, 2000).
         In some cases, resolution varied depending on the application where microchips were used. Tian, Brody, et al. (2000) compared CE-based HDA with microchip electrophoresis in detecting six heterozygous mutations, deletions, and insertions. Microchip electrophoresis succeeded in detecting the mutations, but at a decreased resolution. Conversely, microchip-based HDA successfully located two homoduplexes, suggesting that microdevices may have increased detection efficiency with some mutations. This variation in resolution suggests that microchips may be better suited to detect certain mutations than others.
         Decreased resolution may be traced back to reagent solutions used as well. Separations must be performed in solutions with high salt concentrations to achieve high resolution (Tian, Brody, et al., 2000). High salt concentrations, which are often required in the Polymerase Chain Reaction (PCR), facilitate sample separation. As a result, the level of resolution achieved is increased. Salt solutions, however, can degrade microchip pathways (Tian, Brody, et al., 2000). To ameliorate the problem, Tian et al. (2000) performed HDA microchip electrophoresis on samples diluted with deionized water. The ability to detect mutations in these diluted samples was reduced. In contrast to previous findings, where salt was shown not to affect the resolution in comparison to HDA, products used directly from PCR yielded comparable or better resolutions. These results suggest that channel walls should be modified to protect them against the adverse effects of salt in the samples.

Chip Design Considerations
         The material composition of the microchip can have a large influence on the effectiveness of microchip electrophoresis. Currently, scientists are still experimenting with chips composed of different materials. Microchips usually consist of channels microfabricated or etched on fused glass or silica wafers (S. Liu et al., 2000). Recently, chip design has started to move away from glass or silica substrates, focusing on polymer substrates instead (Chen & Chen, 2000). Such polymers are cheaper and easier to manipulate than glass or silica. One recent study by Chen and Chen (2000) used Plexiglas and a wire-imprinting method to create a chip. This method decreased the variability of channel widths both in the same chip (6%) and between chips (10%). Wire-imprinted chips demonstrated a resolution greater than or equal to that of traditional microfabricated chips.
         Since throughput is directly correlated with the number of separation channels available on a chip (S. Liu et al., 2000), an effective chip layout is critical to the success of the separation. Many variations of microchip design are possible, but the most effective designs incorporate the same features, including effective use of chip space, simultaneous perpendicular scanning by the sample detector across all channels, and uniform injection capability (S. Liu et al., 2000). Depending on the application, different channel shapes may be required to produce effective results. One chip design incorporated right-angle turns in their channels; S. Liu at al. (2000) found that using right angles in the capillary tubes promoted separation that is ideal for analysis of DNA fragment size. These same tubes, however, proved less effective when sequencing separations.
         The most evident restriction in layout is the size of the chip itself. S. Liu et al. (2000) concluded that on average, channel lengths cannot exceed half of the diameter of the chip. On a 10-cm-diameter chip, they achieved separation lengths of 3.3 cm. Although channels of such short length are ideal for restriction fragments, they restrict length of the readings in sequencing separations. Tian, Brody, et al. (2000) concluded that resolution increases with the use of longer separation channels. S. Liu et al. (2000) also found that straight channels should be used for most applications, since curved channels reduce separation resolution. They note that channels should be within scanning range of the detector and should be equal in length from anode to cathode ends to produce electrical field strengths of equal magnitude in all channels.

Conclusion
         Much work is necessary to standardize the materials and equipment used in microchip electrophoresis. As shown by the variations in both chip characteristics and reagent use on resolution, microchips require a degree of calibration and specialization to match each application. While recent experiments in microchip electrophoresis have generated much insight on the variables affecting sample resolution and effectiveness, further experimentation with chip materials and conditions seems likely. Results produced thus far have yet to be integrated into a single consistent, reproducible method.
         Yet the benefits of microchip-based electrophoresis cannot be ignored. Microchip electrophoresis shows great potential over proven slab gel techniques: it is fast, inexpensive, requires minute amounts of sample, and promises automation. To harness the potential of the microchip medium, automated matrix replacement and sample loading should be used (S. Liu et al., 2000). Sensitive automation and channel processing equipment must be incorporated to handle the small sample volumes allowed by microchips. The specifications of such fundamental components, however, are not standard in all microdevices (Schmalzing et al., 2000). Before microchip-based electrophoresis can be embraced, the machinery necessary for sample loading and detection must be standardized.


Literature Cited

Chen, Y., and Chen, S. 2000. Analysis of DNA fragments by microchip electrophoresis fabricated on poly(methyl methacrylate) substrates using a wire-imprinting method. Electrophoresis 21: 165-170.

Liu, S., Ren, H., Gao, Q., Roach, D.J., Loder, R.T., Armstrong, T.M., Mao, Q., Blaga, I., Barker, D.L., and Jovanovich, S.B. 2000. Automated parallel DNA sequencing on multiple channel microchips. PNAS 10: 5369-5374.

Liu, Y., Foote, R.S., Culbertson, C.T., Jacobson, S.C., Ramsey, R.S., and Ramsey, J.M. 2000. Electrophoretic Separation of Proteins on Microchips. J. Microcolumn Separations 12(7): 407-411.

Rodriguez, I., Jin, L.J., and Li, S.F.Y. 2000. High-speed chiral separations on microchip electrophoresis devices. Electrophoresis 21: 211-219.

Schmaizing, D., Belenky, A., Novotny, M.A., Koutny, L., Salas-Solano, O., El-Difrawy, S., Adourian, A., Matsudaira, P., and Ehrlich, D. 2000. Microchip electrophoresis: a method for high-speed SNP detection. Nucleic Acids Research 28: 1-6.

Tian, H., Brody, L.C., and Landers, J.P. 2000. Rapid Detection of Deletion, Insertion, and Substitution Mutations via Heteroduplex Analysis Using Capillary- and Microchip-Based Electrophoresis. Genome Research 10: 1403-1413.

Tian, H., Jaquins-Gerstl, A., Munro, N., Trucco, M., Brody, L.C., and Landers, J.P. 2000. Single Strand Conformation Polymorphism Analysis by Capillary and Microchip Electrophoresis: A Fast, Simple Method for Detection of Common Mutations in BRCA1 and BRCA2. Genomics 63: 25-34.