An Introduction to MEMs Engineering - Nadim Maluf and Kirt Williams Part 11 pptx

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.. DNA Addressing with Mic

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 1µl 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

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

T - A

A - T

G - C

C - G

A A G C

T A G A

G G C T

C G

-G C

-C G

-T - A

C G C A

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 G G A

Fluorescent tag

Unknown strands

in solution

Array bound

to substrate Match No match No match No match 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)

UV light

4 Repeat for other nucleotides

5 Build array until it is 25 nucleotides long

G A

A T

A T

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 425(equal to 1015) different combi-nations that can be made with this process However, with a final chip size of 1.28

cm2, 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 cm2 of electrode area Incidentally, this is precisely the principle of

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

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 potensequen-tially 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

−− −

−− −

−

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 Negaposi-tively 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 1,000µV 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

100 m µ

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.)

[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.

[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).

.

C H A P T E R 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