other DNA detection method—as well as include all fluid preparation and handling functions, such as pumping, valving, filtering, mixing of reagents, and rinsing. This demands the development of a complete system with many enabling technologies, MEMS being only one of them. The electrophoresis part of the DNA sequencing process has been commercial - ized by companies such as Caliper Life Sciences, Inc., of Hopkinton, Massachu - setts, with the product now being sold as the LabChip by Agilent Technologies. Up to 12 samples containing variable-length sections of DNA are placed in the dispos - able LabChip, which is inserted into the Agilent 2100 Bioanalyzer system for analy - sis. This system is about the size of a small suitcase, has a separate computer for control and data acquisition, and is powered by a wall outlet, making the system semiportable. The entire LabChip structure is made of sheets of glass. Patterning of glasses is limited to usually photolithography and etching or laser ablation (see Chapter 3). The layers are bonded together under heat and pressure, then cut apart. The use of glass in a simple process leads to low cost, making a single use before disposal economical. Single-use devices have the advantages of no concern about cross con - tamination from previous samples, greatly reduced chances of clogging, and no long-term risk of material degradation with use. Many glasses (and plastics) are transparent to visible and UV light, which is useful in optical detection schemes. Some specifications for the Agilent DNA 1000 LabChip include a DNA concentra- tion range of 0.5–50 ng/µl, a sizing range of 25–1,000 base pairs, a sizing accuracy of ±15%, and a resolution better than 10% over most of the range [19]. The sample volume is 1 µl and takes 30 min to analyze. DNA Hybridization Arrays Once fragments of an unknown DNA sample have been amplified into many copies, they can be read with DNA hybridization arrays. These are different sequences of preassembled nucleotides attached to a substrate (see Figure 6.7). The DNA sections to be identified, with lengths in the range of a hundred to thousands of bases, are tagged with a fluorescent dye at one end. When placed in a buffer solution on the substrate, sections of some of the unknowns hybridize to the complementary sequences on the substrate. As discussed earlier, hybridization is the process by which DNA strands match up and bind with complementary DNA capture probes. The substrate is then rinsed and illuminated. The locations of fluorescence indicate where hybridization occurred and thus which sequences are present in the unknown. This approach is particularly beneficial in the detection of specific gene mutations and in the search for known pathogens. Several companies commercially produce microscale DNA arrays. One of the market-leading products is the GeneChip  from Affymetrix of Santa Clara, California [20]. The GeneChip is produced on 5-inch square fused quartz substrates, which are coated with a bonding layer comprised of molecules to which the DNA nucleotides can adhere, followed by a protection group [21, 22]. Using a standard photolithographic mask (see Chapter 3), ultraviolet light is shone through 20-µm square openings to remove the protection groups, activating selected sites on the substrate (see Figure 6.8). A solution containing one type of nucleotide (A, T, G, or C) with a removable protection group is flushed across the surface. These 180 MEMS Applications in Life Sciences nucleotides bond to activated sites in each square that was exposed but not in the other areas. The process is repeated to start chains of the other three-nucleotide types. Repeated exposure with different masks to remove the protection groups and flushing with the four nucleotide solutions grow DNA strands, or probes, that are DNA Analysis 181 T-A A-T G-C C-G A A G C T A G A G G C T CG- GC- CG- T-A C G C A F F A T C G F A T C G F A T C G F A T C G F F G C G A F G C G A F G C G A F GCGA Fluorescent tag Unknown strands in solution Array bound to substrate Match Match No match No match No match No match Figure 6.7 The use of a DNA hybridization array. Only complementary DNA fragments in the solution match can hybridize to the fragments bound to the substrate. The free fragments, which are usually much longer than the bound fragments, have fluorescent tags on the end for reading. Only sites that receive their complements will fluoresce when read. 1. Coat substrate with bonding molecules and protection group Protection group Bonding molecule Fused quartz substrate 2. Expose UV light through mask to deprotect exposed area Mask 3. Flush with solution containing one nucleotide (e.g., A) A AA UV light 4. Repeat for other nucleotides A TCGATCGATCG 5. Build array until it is 25 nucleotides long A TCGATCGATCG AT CGC CGCCC AA ATCG CGAA ATCG CCTC CG GA AT AT 25 nucleotides Figure 6.8 Illustration of the GeneChip fabrication process. (After: [20].) typically 25 nucleotides long. Finally, all probes are deprotected, the substrates are diced, and they are packaged in plastic flow-cell cartridges for use. With 25 nucleotides in a sequence, there are 4 25 (equal to 10 15 ) different combi - nations that can be made with this process. However, with a final chip size of 1.28 cm 2 , there is only enough space for about 320,000 squares with different sequences. Thus Affymetrix produces chips with only preselected sequences, targeting specific applications (e.g., detecting strains of E. coli or hereditary neurological disorders in humans). If different sequences or longer lengths are desired, custom arrays can be made either with a new mask set or with a special maskless project system, such as one based on Texas Instruments’ DLP (see Chapter 5), available from BioAutoma - tion of Plano, Texas [21]. Another microarray market leader is Agilent Technologies. One product, the Human 1A Oligo Microarray, has over 18,000 probes per 1- by 3-in glass slide with lengths of 60 nucleotides [23]. Agilent uses inkjet technology (see Chapter 4) to write the probes, base by base, with processing similar to that for the Affymetrix probes. Picoliter volumes of nucleotide “ink” write round spots approximately 130 µm across. In addition to standard products, custom arrays can be produced with a shorter turnaround time than with the masking production method. Agilent also manufactures the Microarray Scanner for reading the arrays and producing com- puter output. The large quantity of data produced by DNA analyses has spawned a new field of study termed bioinformatics, which seeks to develop algorithms to han- dle large genetic databases. Microelectrode Arrays Electrodes are extremely useful in the sensing of biological and electrochemical potentials. In medicine, electrodes are commonly used to measure bioelectric signals generated by muscle or nerve cells. In electrochemistry, electric current from one or many electrodes can significantly alter the properties of a chemical reaction. It is natural that miniaturization of electrodes is sought in these fields, especially for applications where size is important or arrays of electrodes can enable new scientific knowledge. Academic research on microelectrodes abounds. The reader will find a comprehensive review of microelectrodes and their properties in a book chapter by Kovacs [24]. In simple terms, the metal microelectrode is merely an intermediate element that facilitates the transfer of electrons between an electrical circuit and an ionic solution. Two competing chemical processes, oxidation and reduction, determine the equilibrium conditions at the interface between the metal and the ionic solution. Under oxidation, the electrode loses electrons to the solution; reduction is the exact opposite process. In steady state, an equilibrium between these two reactions gives rise to an interfacial space charge region—an area depleted of any mobile charges (electrons or ions)—separating a surface sheet of electrons in the metal electrode from a layer of positive ions in the solution. This is similar to the depletion layer at the junction of a semiconductor p-n diode. The interfacial space charge region is extremely thin, on the order of 0.5 nm, resulting in a large capacitance on the order of 10 -5 F per cm 2 of electrode area. Incidentally, this is precisely the principle of 182 MEMS Applications in Life Sciences operation in electrolytic capacitors. A simple electrical model for the microelec - trode consists of a capacitor in series with a small resistor that reflects the resis - tance of the electrolyte in the vicinity. The fabrication of microelectrode arrays first involves the deposition of an insu - lating layer, typically silicon dioxide, on a silicon substrate (see Figure 6.9). Alterna - tively, an insulating glass substrate is equally suitable. A thin metal film is sputtered or evaporated and then patterned to define the electrical interconnects and elec - trodes. Gold, iridium, and platinum, being very chemically inert, are excellent choices for measuring biopotentials as well as for electrochemistry. Silver is also important in electrochemistry because many published electrochemical potentials are referred to silver/silver-chloride electrode. It should be noted that wire bonding to platinum or iridium is very difficult. If the microelectrode must be made of such metals, it is necessary to deposit an additional layer of gold over the bond pads for wire bonding. The deposition of a silicon nitride layer seals and protects the metal structures. Openings in this layer define the microelectrodes and the bond pads. The following sections describe two instances where microelectrodes show promise as a diagnostics tool in biochemistry and biology. DNA Addressing with Microelectrodes A unique and novel application patented by Nanogen of San Diego, California [25], makes use of microelectrode arrays in the analysis of DNA fragments of unknown sequences. The approach exploits the polar property of DNA molecules to attract them to positively charged microelectrodes in an array. The analysis consists of two sequential operations, beginning first with building an array of known DNA cap- ture probes over the electrode array, followed by hybridization of the unknown DNA fragments. DNA capture probes are synthetic short chains of nucleotides of known specific sequence. Applying a positive voltage to a selection of microelectrodes in the array attracts previously synthesized DNA capture probes to these biased electrodes, where they chemically bind in permeable hydrogel layer that had been impregnated with a cou - pling agent (see Figure 6.10) [26]. Microelectrodes in the array that are negatively biased remain clear. Subsequent washing removes only unbound probes. Immersion Microelectrode Arrays 183 Metal bondpad (e.g., Au) Silicon Silicon oxide Silicon nitride Microelectrode (e.g., Au, Pt, Ir, Ag) C R Figure 6.9 Cross section of a microelectrode array showing two different metals for the elec - trodes and for the bond pads. The schematic also illustrates a basic electrical equivalent circuit that emphasizes the capacitive behavior of a microelectrode. The silicon substrate and the silicon diox - ide dielectric layer may be substituted by an insulating glass substrate. in a second solution binds a second type of DNA capture probes to another set of biased electrodes. Repetition of the cycle with appropriate electrode biasing sequen - tially builds a large array containing tens and potentially hundreds of individually distinct sites of DNA capture probes differing by their sequence of nucleotides. The removal of a capture probe from a particular site, if necessary, is simple, accom - plished by applying a negative potential to the desired microelectrode and releasing the probe back into the solution. It is this electrical addressing scheme to selectively attract or repel DNA molecules that makes this method versatile and powerful. Once the array of DNA capture probes is ready, a sample solution containing DNA fragments of unknown sequence (target DNA) is introduced. These fragments hybridize with the DNA capture probes—in other words, the target DNA binds only to DNA capture probes containing a complementary sequence. Optical imaging of fluorescent tags reveals the hybridized probe sites in the array and, consequently, information on the sequence of nucleotides in the target DNA. This approach is par - ticularly beneficial in the detection of specific gene mutations or in the search for known pathogens. Positive biasing of select electrodes during the hybridization phase accelerates the process by actively steering and concentrating with the applied electric field tar - get DNA molecules onto desired electrodes. Accelerated hybridization occurs in minutes rather than the hours typical of passive hybridization techniques. The 184 MEMS Applications in Life Sciences − − − − − − − DNA capture probe Microelectrode (a) Electronic addressing (b) Detection by hybridization DNA capture probe Target DNA Fluorescent tag A C T G C G A Selected electrode ? ? ? ? ? ? ? ? ? T C C G A G T ? ? Inferred sequence Probe A Probe B Figure 6.10 Illustration of the Nanogen electronic addressing and detection schemes. (a) A posi - tive voltage attracts DNA capture probes to biased microelectrodes. Negatively biased electrodes remain clear of DNA. Repetition of the cycle in different solutions with appropriate electrode bias - ing sequentially builds an array of individually distinct sites of DNA capture probes that differ by their sequence of nucleotides. (b) A DNA fragment with unknown sequence hybridizes with a DNA capture probe with a complementary sequence. Fluorescence microscopy reveals the hybridized site and, consequently, the unknown sequence. method is sufficiently sensitive to detect single base differences and single-point mutations in the DNA sequence. Cell Cultures over Microelectrodes Many types of cells, in particular nerve and heart cells, can grow in an artificial cul - ture over a microelectrode array. The growth normally requires a constant tempera - ture, often at 37ºC (the core temperature of the human body), a suitable flow of oxygen, and a continuous supply of nutrients [27]. Bioelectric activity, or action potential, capacitively couples across the cell membrane and surrounding fluid to the nearest microelectrode, which then measures a small ac potential, typically between 10 and 1,000 µV in peak amplitude. The array of microelectrodes essen - tially images the dynamic electrical activity across a large sheet of living cells. The measured action potentials and their corresponding temporal waveforms are char - acteristic of the cell type and the overall health of the cell culture. For example, tox - ins that block the flow of sodium or potassium ions across the cell membrane suppress the action potentials or alter their frequency content (see Figure 6.11) [27]. This approach may be useful in the future for studying the effects of experimental drugs in vitro or for the early detection of airborne toxic particles. Summary In recent years, a number of microscale biological analysis techniques have become commercialized, notably electrophoresis and arrays for DNA analysis on disposable glass or plastic chips. Prototypes and products to run analyses are becoming smaller and more portable. Most of these biological applications employ microfluidics, in which pumping methods are different than in the macroscopic world and Reynolds numbers are very low. Summary 185 100 mµ Cells Electrode Figure 6.11 Photograph of a cultured syncytium spontaneously beating over a microelectrode array. The platinum electrodes are 10 µm in diameter with a spacing of 100 µm. The electrodes measure the extracellular currents generated by a traveling wave of action potential across the sheet of living cells. (Courtesy of: B. D. DeBusschere of Stanford University, Stanford, California.) References [1] Manz, A., N. Graber, and H. M. Widmer, “Miniaturized Total Chemical Analysis Systems: A Novel Concept for Chemical Sensing,” Sensors and Actuators B, Vol. B1, 1990, pp. 244–248. [2] Kovacs, G. T. A., Micromachined Transducer Sourcebook, Boston, MA: WCB McGraw- Hill, 1998, Section 6.6. [3] Sharp, K. V., et al., “Liquid Flow in Microchannels,” in The MEMS Handbook, M. Gad-el- Hak (ed.), Boca Raton, FL: CRC Press, 2002, Chapter 6. [4] Kopf-Sill, A. R., et al., “Creating a Lab-on-a-Chip with Microfluidic Technologies,” in Inte - grated Microfabricated Biodevices, M. J. Heller and A. Guttman (eds.), New York: Marcel Dekker, 2002, Chapter 2. [5] Gray, B. L., et al., “Novel Interconnection Technologies for Integrated Microfluidic Sys - tems,” Sensors and Actuators A, Vol. 77, 1999, pp. 57–65. [6] Stryer, L., Biochemistry, New York: W. H. Freeman and Co., 1988, pp. 71–90, 120–123. [7] Darnell, J., L. Harvey, and D. Baltimore, Molecular Cell Biology, 2nd ed., New York: Scien - tific American Books, 1990, p. 219. [8] Nguyen, N. -T., and S. T. Wereley, Fundamentals and Applications of Microfluidics, Nor - wood, MA: Artech House, 2002. [9] Mastrangelo, C. H., M. A. Burns, and D. T. Burke, “Microfabricated Devices for Genetic Diagnostics,” Proceedings of the IEEE, Vol. 86, No. 8, August 1998, pp. 1769–1787. [10] Wilding, P., M. A. Shoffner, and L. J. Kricka, Clinical Chemistry, Vol. 40, No. 9, September 1994, pp.1815–1818. [11] U. S. Patent 5,674,742, October 7, 1997. [12] Northrup, M. A., et al., “DNA Amplification with a Microfabricated Reaction Chamber,” Proc. 7th Int. Conf. on Solid-State Sensors and Actuators, Yokohama, Japan, June 7–10, 1993, pp. 924–926. [13] Belgrader, P., et al., “Development of Battery-Powered, Portable Instrumentation for Rapid PCR Analysis,” in Integrated Microfabricated Biodevices, M. J. Heller and A. Guttman (eds.), New York: Marcel Dekker, 2002, Chapter 8. [14] TaqMan ® EZ-RT PCR Kit, Protocol, Applied Biosystems, Foster City, CA, 2002. [15] Kuhr, W. G., and C. A. Monnig, “Capillary Electrophoresis,” Analytical Chemistry, Vol. 64, 1992, pp. 389R–407R. [16] Manz, A., et al., “Planar Chips Technology for Miniaturization and Integration of Separa - tion Techniques into Monitoring Systems. Capillary Electrophoresis on a Chip,” Journal of Chromatography, Vol. 593, 1992, pp. 253–258. [17] Woolley, A. T., and R. A. Mathies, “Ultra-High Speed DNA Sequencing Using Capillary Electrophoresis Chips,” Analytical Chemistry, Vol. 67, 1995, pp. 3676–3680. [18] Woolley, A. T., and R. A. Mathies, “Ultra-High Speed DNA Fragment Separations Using Capillary Array Electrophoresis Chips,” Proceedings of the National Academy of Sciences USA, Vol. 91, November 1994, pp. 11348–11352. [19] Agilent Technologies, Product Literature for DNA1000 LabChip Kit, Palo Alto, CA, 2001. [20] Affymetrix, GeneChip Product Literature, Santa Clara, CA, 2003. [21] Garner, H. R., R. P. Balog, and K. J. Luebke, “Engineering in Genomics,” IEEE Engineer - ing in Medicine and Biology, July/August 2002, pp. 123–125. [22] Fodor, S. P., et al., “Multiplexed Biochemical Assays with Biological Chips,” Nature, Vol. 364, No. 6437, 1993, pp. 555–556. [23] Agilent Technologies, Inc., Product Brochure for Agilent SurePrint Technology, Palo Alto, CA, 2001. 186 MEMS Applications in Life Sciences [24] Kovacs, G. T. A., “Introduction to the Theory, Design, and Modeling of Thin-Film Microe - lectrodes for Neural Interfaces,” in Enabling Technologies for Cultured Neural Net - works, D. A. Stenger and T. M. McKenna (eds.), San Diego, CA: Academic Press, 1994, pp. 121–166. [25] U.S. Patents 5,605,662, February 25, 1997, and 5,632,957, May 27, 1997. [26] Heller, M. J., et al., “Active Microelectronic Array Systems for DNA Hybridization, Geno - typing, Pharmacogenomic, and Nanofabrication Applications,” in Integrated Microfabri - cated Biodevices, M. J. Heller and A. Guttman (eds.), New York: Marcel Dekker, 2002, Chapter 10. [27] Borkholder, D. A., B. D. DeBusschere, and G. T. A. Kovacs, “An Approach to the Classifi - cation of Unknown Biological Agents with Cell Based Sensors,” Tech. Digest Solid-State Sensor and Actuator Workshop, Hilton Head Island, SC, June 8–11, 1998, pp. 178–182. Selected Bibliography Heller, M. J., and A. Guttman (eds.), Integrated Microfabricated Biodevices, New York: Marcel Dekker, 2002. Horton, R. M., and R. C. Tait, Genetic Engineering with PCR, Norfolk, UK: Horizon Press, 1998. The reader will find extensive coverage of the research activities in this field in past proceed - ings of the conference on Micro Total Analysis Systems (µTAS). Summary 187 . CHAPTER 7 MEM Structures and Systems in RF Applications “The discovery of electrical waves has not merely scientific interest though that alone inspired it it has had a profound influence on civilization; it has been instru - mental in providing the methods which may bring all inhabitants of the world within hearing distance of each other and has potentialities social, educational and political which we are only beginning to realize.” —Sir Joseph. J. Thomson, on James Maxwell’s discovery of electromagnetic waves in James Clerk Maxwell: A Commemorative Volume 1831–1931, The University Press: Cambridge, UK, 1931. Radio-frequency (RF) MEM devices have been in research and development for years, with scores of papers published annually. There are unpublicized devices in use in small volume in commercial and military applications, but only recently have such devices gone into high-volume production. Current and future RF MEMS devices will be competitive with more conventional components on the basis of vol- ume, mass, cost, and performance. The largest potential market is in cellular tele- phone handsets, with hundreds of millions of units sold each year. Other portable electronics markets, where the aforementioned qualities are major considerations, include cordless phones for home use, wireless computer networking, radios, and global positioning system (GPS) receivers. Satellites, missile guidance, military radar, and test equipment are separate markets of importance, with lower potential sales volumes but higher unit prices. Opening the cover of a modern cellular telephone reveals a myriad of discrete passive and active components occupying substantial volume and weight. The mar - ket’s continued push for small portable telephones argues a convincing economic case for the miniaturization of components. MEMS technology promises to deliver miniature integrated solutions including variable capacitors, inductors, oscillators, filters, and switches to potentially replace conventional discrete components. Signal Integrity in RF MEMS A requirement for any RF device is maintaining signal integrity: transmitting desired signals with low loss, minimizing reflections, not permitting external signals or noise to join the transmitted signal, and filtering out or not generating undesired signals, such as higher-frequency harmonics. At high frequencies, these seemingly simple requirements are not readily attained. 189 [...]... cost, and thermal coefficient of expansion mismatch factor into substrate selection Passive Electrical Components: Capacitors and Inductors Quality Factor and Parasitics in Passive Components All capacitors and inductors have parasitics associated with them that limit their performance Two parameters that describe their performance and enable comparisons between devices are the quality factor Q and the... self-resonance shortcoming and to reduce the part count and space used on a printed circuit board, low-cost, highperformance on-chip inductors are desirable Example inductor parameters needed for use in an on-chip high-Q resonant tank circuit for VCOs in cellular phones in the 1–2 GHz range are L = 5 nH and Q >30 [4] Inductors are readily fabricated on integrated-circuit chips using standard CMOS or bipolar... one layer of metal and a connection to the center of the spiral in another layer of metal (see Figure 7.4) Losses from the resistance of the metal and eddy currents in the substrate limit the Q to less than 10 at 2 GHz [11] One approach to improving both quality factor and self-resonance frequency is to reduce the parasitic capacitance and substrate conductive loss by changing to an insulating substrate,... series resistance; (c) capacitor with parasitic series inductance; and (d) inductor with parasitic parallel capacitance between coils 192 MEM Structures and Systems in RF Applications frequency range Above the self-resonance frequency f SR =1 / (2π CL para ), the inductance dominates and the capacitor looks to a circuit like an inductor (i.e., the pair has an imaginary positive impedance) Inductors are... on-chip capacitors is the reduction in parts that must be used on a circuit board and the commensurate reduction in cost Other reasons include noise reduction and lowering both parasitic capacitance and resistance Because the capacitance per unit area in a standard process is relatively small, large capacitances (more than a few picofarads) occupy too great a chip area to be cost effective, and high-dielectric... surface-micromachined and bulk-micromachined Surface-micromachined variable capacitors tend to be simpler to fabricate, more readily integrated on the same chip as existing circuitry, and use less expensive process steps than their Passive Electrical Components: Capacitors and Inductors 193 bulk-micromachined counterparts, but they have a nonlinear response to the tuning voltage and smaller tuning ranges... springs [8]; (d) top view using L-shaped springs [5]; and (e) top view with center anchor [7] 194 MEM Structures and Systems in RF Applications An optimal solution is to make the spring beams short, thick, and wide, but within the constraint of the spring constant The resistance can also be kept to a minimum by the use of a highly conductive metal Design and fabrication so that the top plate moves... Interdigitated-finger capacitor: (a) conceptual top view showing a group of fingers and springs; and (b) process flow for reduced substrate parasitics (After: [11] .) 196 MEM Structures and Systems in RF Applications the length of overlap and thus the capacitance between the fingers The capacitance scales linearly with the number of fingers and the finger thickness and is inversely proportional to the gap... mechanical spring constant Another design consideration is that electrical current must flow through the springs to the top plate, making the springs the dominant source of series resistance The geometrical dimensions of the springs can be optimized to provide the least electrical resistance for a particular spring constant Suspended top plate Anchor to Spring substrate Top plate Beam spring Anchor to. .. and the self-resonance frequency fSR The quality factor Q is a measure of loss in a linear-circuit element and is defined as the maximum energy stored during a cycle divided by the energy lost per cycle For reactive components such as capacitors and inductors, it is equal to the absolute value of the ratio of the imaginary part of the impedance to the real part of the impedance: for a capacitor C with . discrete inductors (in addition to even more capacitors and resistors) along with only 15 integrated circuits [12]. To alleviate the self-resonance shortcoming and to reduce the part count and space. of expansion mismatch factor into substrate selection. Passive Electrical Components: Capacitors and Inductors Quality Factor and Parasitics in Passive Components All capacitors and inductors. masks to remove the protection groups and flushing with the four nucleotide solutions grow DNA strands, or probes, that are DNA Analysis 181 T-A A-T G-C C-G A A G C T A G A G G C T CG- GC- CG- T-A C G C A F F A T C G F A T C G F A T C G F A T C G F F G C G A F G C G A F G C G A F GCGA Fluorescent tag Unknown