miniaturization technologies for molecular diagnostics...tors to look beyond the hype of cloning...

Miniaturization Technologies for MolecularDiagnostics

Ronald C. McGlennen

Background: Molecular diagnostics devices are becom-ing smaller. With the advancement of miniaturizationtechnologies, microchip-based systems will soon beavailable for genetic testing. The purpose of this reviewis to highlight the underlying principles in miniaturiza-tion, the strategies being developed for bioanalysis, andthe potential impact on the practice of this rapidlygrowing medical discipline.Approach: The author discusses DNA microchips andtheir practical importation into the clinical laboratory,based on his background in medical device and micro-chip design and development. His discussion is sup-ported by a body of literature covering both biomedicaland electrical engineering and more recent publicationsin the field of molecular genetics and pathology.Content: This review is descriptive and intended tooutline the technologic and methodologic approaches tothe creation of an integrated genetic analysis instrumentbased on miniature components. The review draws onpublished scientific evaluations of these devices with-out regard to the companies involved in their develop-ment.Summary: The intent of this review is that the readerwill better understand the variety of technical ap-proaches toward the miniaturization of molecular ge-netic testing for the clinical laboratory. With insight intothe principles underlying the operation of these chipsand the integrated systems, the end user can betterevaluate the value to the field in terms of makingmolecular genetics testing simpler, faster, and lessexpensive.© 2001 American Association for Clinical Chemistry

Two RevolutionsThere are two revolutions underway in the clinical labo-ratory, and they are both likely to reshape the way we

practice medicine. The first revolution is about how toorganize, innovate, and implement new technologies tomake laboratory testing less costly and less of a burden onthe healthcare system. The second revolution is aboutfinding intelligent ways to make sense out of, and thenapply some of the information we get from genetictesting. For better or worse, molecular diagnostics, adiscipline that uses DNA or RNA combined with molec-ular genetics methods to do diagnostic testing for patientcare, is at the intersection of these two revolutions.

Over the past 10 years, the field of molecular diagnos-tics has experienced phenomenal growth and now repre-sents a $1.2 billion industry. Recognizing this potential, in1997, writers from Fortune magazine admonished inves-tors to look beyond the hype of cloning “Dolly” to theriches to be gained by sequencing the human genome andto place their bets on the exciting new biochip industry.Actually, the potential of miniaturizing molecular diag-nostics was foreseen by those who inspired the HumanGenome Project and thus made one of its primary goalssupport of the invention of new technologies that makethe process of gene discovery faster and less expensive. Byand large this goal has been met: the cost of sequencing aDNA fragment is now 300 times lower than when theprogram started. In July 2000, the rough draft of thesequence of the human genome was completed, demon-strating not only the power of the science, but also thevitality of the private and public collaboration that madethis feat possible. A similar partnership is fueling thebiochip revolution, which will aid in proofreading of thisinitial draft as well as encouraging the development ofuseful applications of this information. At last count, ahandful of large companies and .100 start-up companieswere committed to the commercialization of miniaturizedinstruments for molecular diagnostics, including thermo-cyclers, DNA microarrays, and other types of biosensors.

Reality CheckAs physicians become versed in the use of genetic infor-mation and gain an appreciation of how gene-basedtesting can be used to manage patients, the reliance onmolecular diagnostics is expected to accelerate. However,

Department of Laboratory Medicine and Pathology, University of Minne-sota Medical School, 420 Delaware St. SE, Minneapolis, MN 55455. Fax612-273-6994; e-mail [email protected].

Received April 13, 2000; accepted November 13, 2000.

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unlike other technologies currently used in clinical labo-ratories, molecular diagnostics is highly labor-intensiveand lacks automation and integration of the variousoperational steps (1 ). With the impetus to offer a widervariety in molecular tests, the reality check for the labo-ratory director is how to address mounting clinical needs(market), but at the same time work to reduce costs. Withthe advent of miniaturization technologies, we are well onour way to seeing routine use of biochips as a solution tothis dilemma.

For the most part, these technologies are still verymuch works in progress, and for this reason, the useful-ness of these biochips is limited to applications in theresearch laboratory. Correspondingly, as companies haveevaluated the contentious landscape for patent protection,the rigorous requirements of the Food and Drug Admin-istration, the standards for quality control in clinicaltesting, and the diminishing opportunity for adequate testreimbursement in the molecular diagnostics market,many have shied away from biochips. Like any newgeneration of technologies, however, some biochip-basedtechnologies show greater promise than others. In gen-eral, there are three questions to ask when assessing thepotential of biochips for use in the clinical realm: Do theywork? Do they help? Do they save time and/or money?This review provides some of the background necessaryto answer these questions.

Miniaturized Systems Really Means Only Chip-sizedBiosensors

Reduction in the costs of performing high-throughputDNA sequencing has been a major goal of the HumanGenome Project. This goal inspired many of the “minia-ture systems” that now include such capabilities as DNAsequencing on a chip.

Correspondingly, reports that describe total-analysismicrosystems, or TAS, for laboratory testing are usuallyreferring to a combination of microchip-sized devicesconnected together or integrated on a single substrate(glass, plastic, or for select types of chip-based biosensor,silicon) used for the detection of nucleic acids or anothertype of analyte. Reducing costs is also a driving force inmolecular diagnostics, and it is critical if this platform fortesting is to compete with more conventional diagnosticmethods. However, molecular diagnostics lack the exten-sive automation and system integration found in clinicalchemistry laboratories (1 ). Hence, to appreciate wherecosts can be decreased in the operational schema ofgenetic testing requires an analysis of the whole system,including sample collection (Fig. 1). On the basis ofcurrent methods, sample collection and nucleic acid ex-traction are the most expensive steps involved in genetictesting (2 ). As a practical matter, however, the miniatur-ization of sample handling, involving principles of mi-crofluidics and the controlled storage and delivery ofbiochemical solutions from reservoirs, has provided amore difficult set of engineering problems. Instead, re-search over the last 5–10 years has focused on adaptingprinciples of miniaturization from the field of microelec-tronics to testing of new concepts for localizing biochem-ical interactions on chips the size of postage stamps.Industry leaders hope this strategy will afford the greatestlikelihood of substantially decreasing costs (3 ).

In the effort to create a portable and inexpensive geneamplification and detection system, there has been theneed to converge expertise in molecular biology andengineering. The goal remains to design miniaturizedlaboratory components that can be produced economi-cally, are very small, and have performance superior tomacroscale systems. Microelectromechanical systems

Fig. 1. Operational scheme of a molecular diagnostics laboratory.The scheme takes into consideration the acquisition of the specimen from the patient and the extraction of nucleic acid from the sample. Miniaturization of these stepsremains the greatest challenge. One solution is an extractor chip composed of a silicon chip with minute pillars for sieving whole blood (45). Test setup typically involvessome type of gene amplification chemistry such as PCR or ligase detection reaction. The miniaturized solution is a chip-based thermocycler (46). The major emphasisin development of miniaturization technology is on DNA chips and various types of biosensors. The integration of these components into a single platform device hasbeen demonstrated, principally involving the combination of a thermocycler and microcapillary electrophoresis for detection (47, 48).

394 McGlennen: Miniaturization for Molecular Diagnostics

(MEMS) are one potential solution because they leveragethe economies of scale realized in the silicon processingindustry while permitting the exploration of new princi-ples of “sensing” not possible with conventional methods.The interface between these two disciplines has led tosome exciting new ideas that only in the last few yearshave been realized as devices with potential commercialviability.

MEMS provide a means to make microminiaturizeddevices that do physical work such as pumping, valving,and oscillating; serve as actuators; and sense physicalphenomena (sensors) by processes identical to those usedto fabricate microelectronic chips. “Physical” sensors arethe primary commercial application of MEMS, althoughnew approaches toward combining the mechanical fea-tures of MEMS with biologic materials are laying thefoundation for a whole new class of “biosensors”. MEMSsensors combined with microelectronic circuitry aresometimes referred to as “smart sensor systems”. Smartsystems in turn can be configured into highly portableand inexpensive handheld or benchtop instrumentation.One potential advantage of MEMS devices is their cost ofmanufacture. Batch fabrication enables the production ofthousands of devices at a cost that is only incrementallyhigher than the cost of manufacturing one. In some cases,MEMS-based gene chips are considered disposable, muchlike the plastics and biochemical reagents used in mostlaboratories.

The manufacture of MEMS involves processes com-mon to the manufacture of microelectronic components,such as computer chips, from silicon, including photoli-thography, surface micromachining, and deposition ofspecial materials such as zinc oxide, a piezoelectric com-posite. The microfabrication of a MEMS device is analo-gous to the construction of a house (Fig. 2). Beforeconstruction starts, blueprints of the house need to bedrawn, creating a template designating the position of thewalls and other key elements of that structure. Similarly,the process of photolithography leads to a series ofphotomasks, each outlining one layer of the three-dimen-sional MEMS structure. Each photomask is projected ontothe surface of a silicon wafer, producing a pattern ofshadows and illuminated areas. Photoresists are light-sensitive epoxies used in photolithography to differen-tially protect and deprotect areas of the wafer. Exposureto specific wavelengths of light causes these epoxies tocross-link or “cure”, or in the shadowed areas, becomevulnerable to dissolution by organic solvents. Photoli-thography leaves a footprint for each detail of the struc-ture on the wafer in a sequential placement of materials,much like a bricklayer might first build the foundationwith bricks and cement in preparation for the footings ofthe vertical walls.

Another process in MEMS fabrication is the depositionof thin films. Typically, this involves the use of hightemperatures and mixtures of gases, bathing the surface

Fig. 2. Schematic for the fabrication of a MEMS sensor.Initial steps involve using photolithography to pattern areas where material is to be removed. (Step 1), a trench is created by etching silicon wafer with plasma, a reactiveion beam, or with chemical vapor. (Step 2), the trench is then filled with polysilicon glass (PSG) to protect that area from the subsequent processing, but which willbe sacrificed at the last step. (Step 3), metals or special composite materials, such as thin films of piezoelectric ceramics, are then added to form the sensingelements. (Step 4), these materials are protected by a process of encapsulation with epoxies, called photoresists, or other thin films composed of nitrides or oxidesof silicon. (Step 5), at the final step, the three-dimensional beam structure is formed through a process of chemical etching to remove the sacrificial materials. Theresult is a free-standing beam with electrodes and embedded piezoelectric actuators and sensors.

Clinical Chemistry 47, No. 3, 2001 395

of the silicon wafer for precisely defined times and atprecisely defined pressures. The result is a deposition ofmaterials that have either electrical or structural proper-ties useful to the finished device. Surface micromachininginvolves the use of a variety of techniques, including dryand/or wet etching procedures, to differentially removematerials from the silicon wafer and/or previously de-posited thin films. Micromachining is performed in con-cert with serial photolithography steps to create struc-tures with intricate details, including vertical walls,freestanding beams or diaphragms, conduits, valves, andspecialized surfaces to affix biochemicals, such as nucleicacid probes.

The leveraging of MEMS and silicon processing tech-nologies to create a genetic testing system on a chip isfocused on two broad areas. The first is in the creation ofminiaturized chambers, reservoirs, and conduits in whichto carry out biochemical reactions such as PCR. Thesecond area includes the creation of unique detectiondevices or sensors. Collectively, however, MEMS offerone of the biggest opportunities for the biotechnologyindustry.

DNA Chip TechnologiesAnyone familiar with the method of using a slot blot, dotblot, or reverse blot to “probe” a sample, such as thatderived from PCR, understands the principles of a DNAarray. The placement of a series or array of oligonucleo-tide probes onto solid substrates such as nylon, paper,and polypropylene is an extension of using blottingmethods to localize the signal when DNA fragmentshybridize to their cognate complements. Various chemis-tries have been used to attach DNA probes to filters, butmost involve a carboxyimide coupling using 59-amino-modified nucleosides placed directly on the surface witha mechanical micropipet. Other methods depend on ini-tially modifying the surface with a stable aliphatic polylinker followed by attachment using standard phosphora-midite chemistry (4 ). Microaddressable arrays or DNAchip arrays are a unique combination of technologieswherein microfabrication of silicon or glass is combinedwith novel ways of affixing gene probes to a silicon orglass surface. The origin of this technology is based onresearch by Foder and co-workers (5, 6), who used thetechnique of photolithography for in situ chemical syn-thesis of biochemicals directly on a silicon substrate. Thisso-called light-directed chemical synthesis incorporatesnucleotides modified with photosensitive linkers as thebuilding blocks in serial rounds of probe assembly (7 ).

The process begins when photosensitive linker mole-cules, which include amino acids or hydroxyl-photopro-tected deoxynucleosides, are covalently attached to siliconwafers when pinpoint selected areas are illuminatedthrough a photolithographic mask, which causes thelinkers to be photodeprotected. In the next round ofsynthesis, the linker is reacted with the subsequent nucle-

otide in solution to form the oligonucleotide probe, butonly at the positions exposed to light. The process isrepeated, each time using a different photomask to exposea new geographic position on each of the hundreds ofchips that are replicated across the wafer. After the firstlayer of nucleotides is fixed at each probe location, theprocess is repeated for the second row of nucleotides. Theprocess permits incredible chemical diversity, where only4 3 N chemical steps can produce a complete set of 4Noligonucleotides of length N, or any subset on a singlechip. Depending on the resolution of the photolitho-graphic mask and instrumentation, probe densities ashigh as 106/cm2 can be achieved, although densities 10times lower are more typical (8 ). Current commercialversions of these chips hold up to 20 000–45 000 probessites in a 1.28-cm2 chip area. A recently reported variationof this approach uses precision direction control of a laserin lieu of photolithography to pinpoint the on-chip syn-thesis of oligonucleotide probes (9 ).

Affymetrix Corporation (Santa Clara, CA) uses thistechnology to make their GenechipTM, which was the firstDNA chip to be approved by the Food and Drug Admin-istration for molecular diagnosis of reverse transcriptasegene mutations in HIV. Subsequently, several iterations ofthe Genechip have been configured for various researchand clinical problems, such as mutational analysis of thep53 and BRCA1 genes and detection of single nucleotidepolymorphisms in the cytochrome P450 system of en-zymes. Importantly, the Genechip is only one componentof a large benchtop system that includes a fluidic station,readout chamber, and a computer and software for inter-pretation of the data.

Directional placement of off-chip-synthesized oligonu-cleotide probes by electrostatic attraction is anothermethod of constructing microaddressable array chips(10 ). Manufacture of the Nanogen chip involves fabrica-tion of a dense array of tiny electrodes overlying amolecular permeability layer. A positive electrical biasapplied to the electrode attracts negatively charged oligo-nucleotide probes to a precise location on the chip. Serialapplication of specific probes and differential activation ofthe electrode lead to the eventual assembly of a probearray (11 ).

Genometrix (Woodland, TX) uses off-chip-synthesizedoligonucleotide probes but delivers them onto the chip bymeans of a microjet dispensing system (12 ). A variation ofthis strategy involves the deposition of polymers on thesurface of the chip to embed generic oligonucleotides,called “zip codes”, at a precise location on the surface ofa glass or silicon chip by means of a piezoelectric micro-dispenser (13 ). Through a process that involves the use ofPCR combined with the ligase chain reaction or othergene amplification techniques, the products of amplifica-tion are then hybridized to the prefabricated gene chips,washed, and subjected to readout analysis (14, 15).


signal over noiseThe readout of DNA chips involves detection of a reportermolecule, which typically is incorporated into the geneamplification product (Fig. 3). Incubation of the geneproduct, which is in solution, with the chip leads tohybridization of a segment of the amplicon sequence tothe immobilized probe, ideally with a one-to-one comple-mentarity. Specificity is achieved by optimizing the hy-bridization conditions, which depend mostly on the de-sign of the probe. Interrogation of the hybridized chip bya laser to evoke fluorescence, or a chemical for chemilu-minescence, is detected by a photomultiplier tube or acharge-coupled device, which ascribes the signal at aparticular location on the array to the known sequence ofthe probe (16, 17). Knowing the pattern of product hy-bridization and the exact sequence of each probe, from thetime when the chip was constructed, permits one todeduce the DNA sequence of the sample being tested(18, 19).

In practice, however, routine use of these chips can bedifficult. Principally, the problems relate to achieving areliable signal-to-noise relationship for each hybrid du-plex sequence. This is more complicated when chips areused that involve the hybridization of products from amultiplex of amplification reactions containing numerous

potential single-nucleotide differences. In fact, difficultiesin interpreting fluorescence-based chips have led manyinvestigators to use a manual readout scheme in whichthe chip is examined through a fluorescence microscopeinstead of with an automated chip reader and associatedsoftware system (20 ). However, turning to manual over-ride is not possible with some of the recent chip con-structs, where as many as 25 000–48 000 independentgene sequences are analyzed simultaneously.

At the molecular level, many chemical as well asphysical factors influence the “quality” of the hybridiza-tion reaction, including steric hindrance of the fluoro-chrome, errors in or degradation of the probe sequence,and orientation of the probe against the surface of the chip(21 ). For these reasons, the development of zip codes hasadvantages in that each probe is designed to have closelymatched physical characteristics and nearly uniformchemical properties, such as melting temperature. Theresult is markedly improved signal-to-noise discrimina-tion and easy interpretation of the data. Moreover,Witowski et al. (22 ) have shown that by creating lamina-tions of zip code probes embedded into acrylamide andacrylamide co-polymer, three-dimensional spots can becreated that increase the probe density and hence theintensity of the fluorescence at a single point.

Fig. 3. Protocol for the use of a DNA microarray.(Step 1), amplified DNA is internally or secondarily labeled with a fluorescent or chemiluminescent reporter. (Step 2), the duplex amplicons are separated into singlestrands by heat or chemicals. (Step 3), the prefabricated DNA array is incubated with the single-stranded sample and hybridization occurs. Like any hybridizationreaction, nonspecific interactions may occur. (Step 4), the hybridized chip is washed using stringency that is empirically determined. (Step 5), the readout of the chip,typically involving fluorescence detection, is done with an automated instrument or a optical microscope. Determination of the DNA sequence or mutation detectionis based on knowing the location and sequence of each probe in the array.


Little Chips, Big BoxesIn each of the above examples as well as in other versionsof microaddressable array technologies, the reliance on anoptical detection system to readout the DNA chip neces-sitates using large and expensive instrumentation. Forthis reason, the primary experience with this technologyhas been in large research facilities and for applications indrug discovery where high throughput and a high outputof information are paramount (23 ). The successful appli-cation of DNA microarrays for gene discovery and cDNAexpression arrays for disease profiling is in fact drivingthe direction of the technical refinement of these chipstoward ones that contain more sequences and requiremore powerful and sophisticated analytic capabilities.These super chips are very expensive, and the big boxesthat read them are increasingly out of the price range ofmost hospital-based laboratories, making the utility ofthese devices in the clinical arena and at the point of careseem more remote than when they were introduced.

A promising handheld DNA analyzer developed byClinical Microsensors (Pasadena, CA) eliminates the needfor an optical readout by sensing the change in electricalimpedance when a DNA substrate is bound to a comple-mentary probe compared with an unbound probe. In thiscase, the DNA probes are attached to electrode pads onthe “chip” through molecular wires (made of a phenyl-acetylene polymer) (24 ). After hybridization of an unam-plified DNA sample to the probe, DNA linked to aferrocene redox label is added. When voltage is applied tothat particular electrode, charge is conducted through theduplex DNA molecule and will have lower impedance,which is measured by the difference in voltage betweenbound and unbound probe electrodes (25 ).

Microfabricated Genetic DevicesOne of the first demonstrations of a microfabricationtechnology for laboratory testing is a tiny microcapillaryelectrophoresis chip. A chip-based system is created usingthe technique of surface micromachining on glass, plastic,or silicon, etching a capillary up to 2 m in length in a spaceno larger than that of a postage stamp. At each end of themicrocapillary is a fluid reservoir. Application of a volt-age across these reservoirs will cause fluid to flow alongthe length of the microcapillary. Analytes, such as dis-solved DNA fragments of various lengths, will separateaccording to their electrophoretic mobility countered bythe oppositely directed electroosmotic flow. Additionalreservoirs, connected by intersecting microcapillaries,permit directional flow of the solution and hence process-ing of specific analytes to their respective “chemicalstations”. Caliper Technologies (Palo Alto, CA) incorpo-rates this differential “electrokinetic flow” into their Labon a ChipTM devices (26–28). A simpler version of amicrocapillary electrophoresis system has been evaluatedfor several genetic markers, including bcr-abl gene tran-scripts associated with chronic myelogenous leukemiaand separation of PCR fragments generated in the factor V

Leiden mutation assay, as well as a multiplexed reactioninvolving mutational analysis of the cystic fibrosis gene(29–31). In each case, the clear advantage of microcapil-lary electrophoresis is its high throughput where theseparation times are on the order of seconds from the timeof sample application.

True MEMS sensors have only recently been shown toalso function as biochips, although none have been dem-onstrated commercially for clinical applications. In alarger sense, MEMS devices take advantage of certainmechanical properties of structures that have homologs inthe macroscale world. One such device is a so-calledquartz microbalance, in which bulk monocrystallinequartz or a microfabricated cantilever-like structure iscombined biochemically with certain probes of knownmass (25, 32, 33). Quartz, having piezoelectric properties,can be put into a high-frequency oscillatory mode wherethe frequency of this oscillation is dependent on the massof the structure. In this case, the DNA probe serves as areceptor that then hybridizes to its cognate DNA tem-plate, producing a structure with a mass larger than thoseof devices not containing bound DNA (34 ). The resultingdevice has a resonant frequency that is shifted downwardor lower than those not displaying specific strand hybrid-ization. A quartz microbalance array has been constructedand has been demonstrated to show high sensitivity ofDNA hybridization to as little as 10218 moles, or ;10212 gof target DNA (35 ).

Other researchers who constructed a surface acousticwave sensor that uses thin-film piezoelectric materialsexploited variations on this theme (36 ). In this case, DNAprobes or other biomolecules immobilized on a planarsurface are set into oscillatory mode by virtue of amicrofabricated transmitter. On the opposite side is amicrofabricated receiver. An oscillatory signal is broad-cast across the piezoelectric field and is detected at thereceiver at a known time, which is dependent on anyimpedance of that transmitted signal, such as that causedby bound DNA. Wang et al. (37 ) demonstrated the utilityof the so-called flexural plate wave sensor for the detec-tion of serum-based proteins through their affinity to amonoclonal antibody affixed to the piezoelectric sensorsurface. McGlennen and co-workers have demonstratedhow other types of piezoelectric thin films can be appliedto structures such as microfabricated diaphragms and/ormicrocantilevers that detect mass changes with DNAhybridization (unpublished results; Fig. 4). In this case,the microcantilever is set into a fixed oscillatory mode thatis altered when hybridization between PCR-derived DNAand its cognate oligonucleotide probe takes place (Fig. 5).These devices are sensitive to picograms of mass loadingbut demonstrate the added advantage of compatibilitywith on-chip microelectronic circuitry. One applicationdemonstrating the potential for clinical use has been thedetection of bacterial contaminants in milk (38 ).

Taken in total, these sensor systems can achieve highsensitivities not observed previously with addressable


array-based approaches and in turn can be linked directlyto microelectronic circuitry, producing a device that ishighly compact and very inexpensive to manufacture(39 ). One proposed device involves a MEMS microcanti-

lever array for gene-based testing, illustrating how detec-tion of mass loading by a shift in resonant frequency ofseveral DNA products has the potential for enhanceddiagnostic sensitivity. Moreover, the commercialization ofMEMS-based sensors offers the promise of lower manu-facturing costs, where individual devices can cost lessthan $1.00.

Surface plasmon resonance is yet another attractivetechnique to directly detect unamplified DNA samples.Surface plasmon resonance is based on intermolecularchanges in the refractive index at the surface of the sensorover time (40 ). In this technique, a DNA template, unam-plified or amplified, is hybridized in a small reservoircontaining an optical interface bound to specific oligonu-cleotide DNA. Through the process of complimentary-based hybridization, light passing through the sensor basewill be refracted at an angle greater than in those sensorsnot demonstrating specific hybridization (41 ). This pro-cess can be calibrated by varying the temperature and issomewhat dependent on time. The specific conditions andkinetics of this interaction, in turn, are characteristic of thedegree of sequence complementarity between the DNAtemplate and the cognate probe. Work by Nilsson et al.(42 ) has demonstrated the utility of surface plasmonresonance in the detection of clinical samples amplified byPCR for a series of clinically relevant gene markers,including p53.

Changes in the optical pathlength have also beendemonstrated to be an effective means of showing DNAhybridization on a silicon microchip interferometer (43 ).An interferometer is a device consisting of an array ofmicrosized pillars or finger-like projections that are cre-ated by etching deep into a silicon wafer. Incident lightshown onto this area of micropillars will be reflectedbackward at a predictable angle of efferent light. Whenthe effective thickness of an individual pillar is increased

Fig. 4. Diagram of a microcantilever biosensor.The MEMS component comprises a microcantilever beam constructed fromsilicon nitride. Overlying this basic beam is a series of layers that include abottom electrode, an insulator, and a top electrode. In this example, a thin filmof the piezoelectric composite material, lead zirconium titanate (PZT), is depos-ited, patterned, and bonded to the electrode stack. The piezoelectric film servestwo functions. One set of piezoelectric strips actuates the beam to oscillate upand down when an AC voltage is applied. A second piezoelectric element sensesthat motion or any changes in the oscillatory frequency. Overlying the piezoelec-tric stack is the biomolecular recognition surface. This layer is composed ofeither a polymer or a self-assembling monolayer containing an embedded probe(an example using an oligonucleotide is described in the text). When a comple-mentary sequence (such as that generated by PCR) is hybridized to the surfaceprobes, the interposed polymer undergoes a conformational change and impartsa bending force to the beam. That force in combination with the added mass ofthe bound DNA is detected by the sensing piezoelectric element as a change inthe resonant oscillatory frequency and is transduced into a measurable voltage.

Fig. 5. MEMS piezoelectric microcantilever sensor.(A), photomicrograph of a piezoelectric microcantilever sensor. The dimensions of the device in this example are 100 mm 3 75 mm, and the beam is 1.5 mm thick.The lateral strips are the actuator elements, and the center strip is the sensor element. (B), output of the oscillating biosensor. The resonance signals produced bythe unbound (peak on the right) and bound (peak on the left) probes are shown.


by coating with DNA probe and subsequent hybridiza-tion of product to the DNA probe, there is a change in theeffective pathlength of the light passing through thecolumn, creating what are called Perot-Fabry fringes. Theshift in the angle of the efferent light is proportional to thequantity of DNA hybridized to the affixed probe. Thus,the technology is highly specific because of the comple-mentarity of DNA-DNA and may also show promise forquantitative analysis.

Each of the above approaches in some way involvesexploitation of either physical properties inherent to DNAor the capability of light-based detection systems toresolve isolated areas of DNA hybridization. Anothersensor system involves the combination of complex sim-ulations of biological systems confined to a silicon micro-chip. These devices are generating great excitement be-cause they appear to be a truly universal platform forbiosensors of the future.

A biosensor that uses an artificial ion channel wasreported by Cornell et al. (44 ). This device takes advan-tage of the sensitive discrimination of naturally occurringreceptor systems and the resolution of natural and artifi-cial biomembranes to function as a transducer and am-plify signals in a highly compact and reproducible sys-tem. Briefly, a lipid bilayer is constructed that contains aknown quantity of the artificial antibiotic gramicidin.Gramicidin exists in a homodimeric form wherein bothmonomers of the protein reside in a linear conformation.Intact homodimeric gramicidin permits the flow of cat-

ions across the lipid membrane through a central pore. Tofunction as a biosensor, one subunit of gramicidin iscovalently linked to a specific cellular receptor, such asthyroid-releasing hormone receptor, or a hapten directedagainst some analyte. Another hapten is tethered anddirected to the lipid membrane. When a ligand, such asthyroid-releasing hormone, binds to its cognate recep-tor, there is competition between the tethered haptenand the one linked to gramicidin. The result is adisruption in the formation and continuity of the gram-icidin transmembrane pores with a net loss of posi-tively charged ions flowing across the membrane. Thischange in current can be detected by a galvanometer,which measures the absolute loss or decrease in currentflow and additionally can record the phenomenon as afunction of time.

The opportunity to use any of the biosensors listedabove is indeed great. By design, many of these ap-proaches can be adapted to a variety of test types, andeach has the promise of high sensitivity and specificity(Table 1). The possibility for miniaturization is also ap-parent, but the commercial application of these for clinicalgene-based diagnostics remains to be realized. The great-est challenges, however, lie in the integration of sensingtechnologies with those “front-end events” such as sam-ple preparation and, in the case of PCR, thermocycler-based assays. Companies such as Cepheid have focusedon technologies to miniaturize blood separation and DNApurification using chips by fabricating a mechanical sieve

Table 1. Comparison of miniaturized sensing technologies.Type of biochip Technical principle Development status

Microcapillary electrophoresis Separation of analytes by electrophoresis in a microfabricatedcapillary 1–30 mm in diameter

Commercialized by Cepheid and CaliperTechnologies, and SoaneTechnologies

DNA hybridization chip Synthesis or site-directed fixation of oligonucleotide probesonto chip surface; detection by laser-induced fluorescence ofhybridized PCR product

Commercialized by Affymetrix forseveral diagnostic tests includingHIV, p53, and BRCA1; othermanufacturers are involved inresearch markets or awaiting FDAa

approvalSurface acoustic wave

sensor (SAW)Detection of change in the propagation of a surface wave from

transmitter to receiver through a molecular binding domainResearch device developed at the

University of California, BerkeleySurface plasmon resonance

(SPR)Detection of the evanescent wave propagated from the

interface of a reflecting metal to which is bound a hybridizedanalyte

Benchtop instrument commercializedby Biacore; microchip-based versiondeveloped by University of Minnesota

Electromechanical MEMSsensor

Detection of mass loading, either by shift in resonantfrequency or by static flex of a piezoelectric transducer

Bioanalytic Microsystems, University ofMinnesota

Porous silicon interferometer Silicon is “roughened” to increase the surface area;oligonucleotide probes are fixed to the surface; lightreflected from the surface will shift in wavelength as afunction of the change in the optical thickness attributableto sample binding

Research development at severalcenters

Artificial biomembrane sensor A phospholipid bilayer containing a synthetic protein, e.g.,gramacidin, over a gold electrode creates ionic pores thatpermit a current to cross the membrane; distortion of thepores because of binding of a ligand to a “tethered”receptor decreases the ionic current, which is measured bya galvanometer

Developed commercially by DunlopCorporation

a FDA, Food and Drug Administration.


and combining this extractor chip with a chip-basedreaction chamber for thermocycling.

Integration Is the KeyReduction of cost is the major concern in laboratorymedicine, and nowhere is that truer than in moleculardiagnostics. To appreciate how chip technology and min-iaturization in general can impact the cost of moleculardiagnostics requires a thorough examination of each ofthe steps involved in performing that test. On the basis ofcurrently available technology, molecular testing is at besta collection of piecemeal operational steps, each of whichis largely manual, and requires expert personnel to per-form tests accurately and reliably. Currently, the “hows”and “whys” of specimen collection, DNA extraction,various methods for nucleic acid amplification, and thechoice of analytic techniques for the products of thoseamplifications are each being worked on as individualpieces of technology, some of them chip-based and othersnot. Unlike disciplines such as clinical chemistry, wherehighly refined and automated instrumentation is com-monplace, molecular diagnostics methods boast little orno automation or system integration. Luckily, moleculardiagnostics will be the first to benefit from recent discov-eries in biochip technology and the convergence of theseemingly unrelated fields of electrical and mechanicalengineering and material science with the breakthroughin commercialization of molecular biology reagents. Inno-vators in the biotechnology industry are realizing that thepath taken to the mass production of computer chips mayalso lead to miniaturized laboratory chips, with the com-mensurate performance, integration, and cost savingsneeded for gene testing to fulfill its potential in clinicalpractice. So promising are the prospects for chip technol-ogy in the diagnostic laboratory that one outcome will berealization of instruments for point-of-care or, possibly,at-home gene testing.

ConclusionAs is often the case with breakthrough technologies, thereis a great deal of drama surrounding the prospect ofcreating micrometer- and even nanometer-scale chips,known as biochips. However, in this case, much of thisoptimism is justified, considering that in just a few shortyears basic research has led to several products that are inor near the commercial phase of their development.Developers of DNA chips believe that in the near futurethese technologies will enable clinicians, and in somecases patients themselves, to quickly and inexpensivelydetect a wide variety of genetic-based diseases and con-ditions, including AIDS, Alzheimer disease, cystic fibro-sis, and several forms of cancer. In other arenas, thistechnology will also make it possible to develop inexpen-sive strategies for screening of new pharmaceutical agentsas well as new genes associated with hitherto uncharac-terized diseases. Until then, however, considerable workneeds to be done to make these technologies useful and

robust as well as inexpensive for the end user. To date, afew companies have commercialized DNA chip products,but many more underlying scientific principles remain tobe discovered before these devices are ready for routineclinical use.

The author is president of Access Genetics, Inc.

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miniaturization technologies for molecular diagnostics...tors to look beyond the hype of cloning...

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