Contains table of contents for Part IV, table of contents for Section 1, and reports on two research projects.Genometrix, Inc. Contract GMX-GH00776-04Defense Advanced Research Projects AgencyU.S. Air Force - Office of Scientific Researc

Adourian, Aram

Chiou, Jeffrey T.

Ehrlich, Daniel J.

Hollis, Mark A.

Koutney, Lance

Matsudaira, Paul T.

Rathman, Dennis D.

Schmaltzing, Dieter

Srivastava, Alok K.

Young, Albert M.

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BioelectronicsSection 1 Genetic Analysis329----- ---- ~---~--~---- -1- -----Part IV.330 RLE Progress Report Number 139Genetic AnalysisChapter 1 Genosensor Technology Development331Section 1332 RLE Progress Report Number 139Chapter 1. Genosensor Technology DevelopmentChapter 1. Genosensor Technology DevelopmentAcademic and Research StaffProfessor Paul T. Matsudaira, Dr. Aram Adouria1 , Dr. Daniel J. Ehrlich, Dr. Mark A. Hollis, Dr. LanceKoutney 1, Dr. Dennis D. Rathman, Dr. Dieter Schmaltzing1 , Dr. Albert M. Young2Graduate StudentsJeffrey T. Chiou, Alok K. Srivastava1.1 Microdetection Technology forAutomated DNA SequencingSponsorGenometrix, Inc.Contract GMX-GH00776-04Project StaffDr. Dennis D. Rathman, Dr. Mark A. HollisThe overall objective of this cooperative work withGenometrix is to develop a novel method for auto-mated, low-cost, high-throughput DNA sequenceanalysis. The overall goal is to demonstrate labora-tory prototypes that provide a substantial increasein speed over the conventional DNA sequencingmethods now used in the biomedical, pharmaceu-tical, and agricultural industries.The basic approach being taken is depicted infigure 1. In a hypothetical DNA sequencing test, asolution of single-stranded "target" DNA strands ofidentical but unknown sequence is washed onto aspecialized microelectronic chip called a geno-sensor. The genosensor surface contains a largearray of test sites, each site containing short piecesof single-stranded DNA known as "probes." Theseprobes are chemically attached to the site. Allprobes in a given site are of like sequence, and thesequence for each site is unique on the chip. Thetarget DNA strands will bond, or hybridize, verystrongly to probes containing their exact Watson-Crick complement, but much less strongly to probeson other sites. The sites containing hybridized DNAare identified via electronic sensing on the chip, andthis information is used by off-chip instrumentationto reconstruct the sequence of the target strands.MIT's role in this effort is to perform the design andfabrication of the genosensor chips.The simplest electrical measurement that can bemade at a test site to detect hybridization is prob-ably a measurement of the change in local permit-tivity due to the addition of long target strands tothe site. The complex permittivity E'-jE" of anaqueous solution containing DNA exhibits a disper-sion around a relaxation frequency which is a func-tion of the size and conformation of the DNAmolecule. A measurement of the capacitanceand/or conductance between two electrodes in thesolution over a range of frequency can therefore dif-ferentiate between a site that contains only shortprobe strands and one that contains long targetstrands hybridized to the probe strands. Fromthese measurements the relative permittivity E', thedielectric loss E" and the dissipation factor E"/E' canbe obtained for the cell.The ideal electrode structure in a test well consistsof two parallel plates spaced so that the entirevolume between them is filled by the hybridizedDNA globules in aqueous solution. For the sizes oftarget DNA envisioned, this spacing ranges fromapproximately 200 to a few thousand angstroms. Apractical, easily fabricated structure that approxi-mates this ideal is the interdigitated design shownin figure 2. Fabricated by a combination of wet anddry etching with metal liftoff, this design canachieve the required spacings between the upperand lower electrodes at their edges. Deviceshaving either Au or Pt electrodes have been fabri-cated, with Pt preferred for most applications.Figure 3 shows various aspects of completed geno-sensor devices.The primary emphasis of our work this past yearwas to optimize the electrode geometry for1 MIT Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.2 MIT Lincoln Laboratory, Bedford, Massachusetts.333Chapter 1. Genosensor Technology DevelopmentFigure 1. Conceptual genosensor system.DETECTION-ELEMENT CROSS SECTIONI- 2 ,r -TI Au AQUEOUS DNA\1. SOLUTIONsi N UNHYBRIDIZEDs34o S DNA PROBESS;012AuSI SUBSTRATEFigure 2. Electrode design for a permittivity genosensor.The unit cell shown is repeated many times across a testwell to form an interdigitated test structure with the topAu electrodes connected to one access line and thebottom Au electrodes to the other.maximum sensitivity. Initial work for this contracthad begun with the electrode configurations illus-trated in figures 2 and 3. Subsequently, bothexperimental studies and theoretical modellingshowed a need to design and fabricate moreaggressive electrode geometries. These have nowbeen developed, as described below.We have now successfully developed the tech-nology for an aggressive self-aligned or split-architecture design using the same Ti-Pt electrodematerials as in previous conventional genosensordesigns. These new devices feature submicron-electrode spacings in both the vertical and hori-zontal dimensions. The patterning of the electrodesis being done by a combination of laser interfer-ometric lithography and reactive ion etching. Ascanning electron micrograph of one of thesedevices is shown in figure 4. The electrode line-width and spacing is 160 nm and the vertical sepa-ration of the interdigitated electrodes isapproximately 200 nm. This geometry yields a 12.5times improvement in the electrode density per unitdevice area. The bodies of the fingers themselvesare Si3N4 to a depth of 200 nm, where thermaloxide is exposed. The use of oxide at the fingerbottoms enables a slight undercut to be obtained bywet etching of the underlying oxide. It is anticipatedthat linker chemistries, designed to attach either tothe closely-packed Pt electrodes or to the sup-porting dielectric posts, will enable a substantialincrease in probe density at the sensor site, with acorresponding increase in detection sensitivity.The development of this submicron self-alignedgenosensor was enabled by successful resolutionof three significant processing challenges: (1)excessive interelectrode metal shorts, (2) highinterconnect-lead resistance, and (3) adaptation ofour insulated-lead process to devices with sub-micron interelectrode spacings.For the conventional 2-micron self-aligned geno-sensor geometries (shown in figures 2 and 3c),interelectrode shorts (connecting top and bottom)have been relatively rare due to the clean sepa-ration of metals resulting from the extensive oxide334 RLE Progress Report Number 139GENOSENSOR SYSTEM)SENSOR INSTRUMENTATION COMPUTER"AGTCG"Chapter 1. Genosensor Technology Development(a)100 pm(c)Figure 3. Collection of photographs showing various views of a complete genosensor device. (a) Top view of thestandard 100 pm X 100 pm single test cell. (b) Top view of the 6 X 6 passive array of test cells. (c) Scanning electronmicrograph (SEM) closeup of the interdigitated electrode fingers. (d) Genosensor chip in electronic package.undercutting inherent in the design. For thesubmicron-based devices, interelectrode shortshave been the leading cause of poor device yield.Because the grating etch is anisotropic andproduces rather straight sidewalls, the sidewalls canend up being coated with a very thin metal layerafter electron-beam deposition of the electrodes,resulting in an interelectrode short. From ameasurement-sensitivity point of view, one wouldprefer to have the electrode layers relatively thickwith minimal vertical and horizontal separation;however, this exacerbates the interelectrodeshorting problem. Thus device yield and devicesensitivity represent a fundamental tradeoff indevice design and fabrication. We have encount-ered this problem in an array of other electronicdevices which employ the same type of gratingarchitecture. Two solutions to this problem that335(d)Figure 4.ture.Im 2A5.. . . ..New submicron-periodicity genosensor struc-(b)Chapter 1. Genosensor Technology Developmenthave been successfully employed in the past arethe use of controlled wet-chemical "dip" etches toremove the thin sidewall metal while leaving mostof the active electrode intact, and/or the use ofelectrical probing to pass small amounts of currentthrough the short to blow open the shorting link.Both techniques have their advantages and disad-vantages. The primary advantage of the electricalunshorting method is that it can also serve as adiagnostic (in some instances) to indicate where theshorting problems are occurring. It should also bepointed out that the device remains functional pro-vided most of the other grating fingers remainintact. In order to prevent the shorts from occurringto begin with, we have improved the process yieldby two process alterations: (1) by increasing theamount of oxide undercut, and (2) by the use ofshallow glancing-angle metal evaporations whichare used to form a metal cap on the grating-fingertops. The net result of these two process improve-ments is illustrated by the SEM shown in figure 5.Note how the metal cap on the finger tops and theenhanced oxide undercutting serve to enhance theseparation of the top and bottom metal electrodes.Typical short-related active-site failures have beenreduced from 90 percent to approximately 10-15percent with these process improvements, and thefew remaining shorted devices are more easily fixedby nondestructive electrical probing.Typical electrode metal thicknesses for theconventional-geometry genosensors were approxi-mately 2000-2500 A in thickness. Thus, the Pt-based interconnect lines were also of the samethickness. However, the submicron-period devicesnecessitate the use of 500-700 A-thick metal elec-trodes for the device active areas. Thus, an addi-tional metallization step is required for thesedevices in order to minimize the interconnect-leadresistance. The liftoff of thick Pt lines for the deviceinterconnects is nontrivial, as has been detailed inpast reports, but previous process development hasenabled us to easily adapt this additional processwith little or no consequence to device yield.A somewhat more difficult task was the incorpo-ration of the insulated-lead process developed ori-ginally for the conventional-geometry devices toeliminate spurious interconnect-associated signals.As described in previous reports, a process wasdeveloped to cover the long Pt interconnect lineswith Si0 2, with subsequent exposure of the deviceactive electrodes done by wet etching of the oxidein windows. This process was enabled in part bythe large finger geometries of the conventionalFigure 5. Closeup of new submicron-periodicity geno-sensor structure showing enhanced undercutting for min-imization of metal-metal shorts.devices which permitted some modest amount ofoveretching. It is clear, however, from thesubmicron-period device shown in figure 5 (note thevery thin bottoms of the grating-finger posts whichare Si0 2) that any overetch will likely result in eitherthe complete collapse of the grating fingers or liftingof the metal-electrode lines off the surface. Duringthis past year, we developed a two-step wetetch/dry etch process which enabled the removal ofthe oxide layer from the active-area electrodes.Our process improvements resulted in an increasein overall yield from roughly 5-10 percent to over 90percent. In all, ten device chips (containing 36active sites each) of the submicron-period designwere successfully fabricated for Genometrix.1.2 Microfluidic Biodevices andApplications of Three-DimensionalLaser Processing to BiodevicesSponsorsDefense Advanced Research Projects AgencyU.S. Air Force - Office of Scientific ResearchProject StaffDr. Daniel J. Ehrlich, Dr. Albert M. Young, Pro-fessor Paul T. Matsudaira, Dr. Aram Adourian, Dr.Lance Koutney, Dr. Dieter Schmaltzing, Jeffrey T.Chiou, Alok K. Srivastava336 RLE Progress Report Number 1390.1 urm1.2.1 Program SummaryThis is a collaborative project between theResearch Laboratory for Electronics and theWhitehead Institute for Biomedical Research todevelop microelectromechanical systems forbiochemical analysis (BioMEMS). The mainemphasis is on developing microfabricated fluidicdevices for sequencing and typing of DNA andprotein. In this program, we invent new microfluidicdevices and develop new microfabrication methodssuch as lithography, glass bonding, and moldedpolymer replication for applications in genetics andcell biology. Our program engineers advancedautomation and optical sensors to supportBioMEMS development.Chapter 1. Genosensor Technology DevelopmentSupported by funding from the National Institutes ofHealth, the Microfluidic Biodevices program installsnew methods of high-volume DNA sequencing atWhitehead's Center for Genome Research.Funding from the Defense Advanced ResearchProjects Agency and the U.S. Air Force's Office ofScientific Research supports advanced applicationsin Department of Defense forensics and detectionof pathological microbes.1.2.2 PublicationYoung, A., and D.J. Ehrlich. "Quantitative Injectionof 100-picoliter Samples for Capillary and Micro-chip Electrophoresis." Submitted to J. Anal.Chem.337338 RLE Progress Report Number 139

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