Part IV Bioelectrordes
SSection 1 Genetic Analysis
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324
RLE Progress Report Number 137
Chapter 1. Genosensor Technology Development
Chapter 1. Genosensor Technology Development
Academic and Research Staff
Dr. Daniel J. Ehrlich, Dr. Mark A. Hollis, Dr. Dennis D. Rathman
1.1 Introduction
The primary objective of this cooperative work with
the Houston Advanced Research Center (HARC) is
to develop a novel method for automated, low-cost,
high-throughput DNA sequence analysis.
The
overall goal is to demonstrate laboratory prototypes
that provide a substantial increase in speed over
the conventional DNA sequencing methods now
used in the biomedical, pharmaceutical, and agricultural industries.
The basic approach is shown in figure 1. In a
hypothetical DNA sequencing test, a solution of
single-stranded "target" DNA strands of identical but
unknown sequence is washed onto a specialized
microelectronic chip called a genosensor.
The
genosensor surface contains a large array of test
sites, each site containing short pieces of singlestranded DNA known as "probes." These probes
are chemically attached to the site. All probes in a
given site have the same sequence, and the
sequence for each site is unique on the chip. The
target DNA strands will bond, or hybridize, very
strongly to probes containing their exact WatsonCrick complement, but much less strongly to probes
on other sites. The sites containing hybridized DNA
are identified via electronic sensing on the chip, and
this information is used by off-chip instrumentation
to reconstruct the sequence of the target strands.
MIT's role in this effort is primarily in the design and
fabrication of the genosensor chips.
1.2 Development of Genosensor Arrays
1.2.1 Genosensor Electronic-Detection
Principle
The simplest electrical measurement that can be
made at a test site to detect hybridization is probably a measurement of the change in local permittivity due to the addition of long target strands to
the site.
The complex permittivity E'-jE" of an
aqueous solution containing DNA exhibits a dispersion around a relaxation frequency which is a function of the size and conformation of the DNA
molecule. A measurement of the capacitance
and/or conductance between two electrodes in the
solution over a range of frequency can therefore differentiate between a site that contains only short
probe strands and one that contains long target
strands hybridized to the probe strands. From
these measurements the relative permittivity E', the
dielectric loss E", and the dissipation factor E"'E' can
be obtained for the cell.
The ideal electrode structure in a test well consists
of two parallel plates spaced so that the entire
volume between them is filled by the hybridized
DNA globules in aqueous solution. For the sizes of
target DNA envisioned, this spacing ranges from
approximately 200 to a few thousand angstroms. A
practical, easily fabricated structure that approximates this ideal is an interdigitated design shown in
figure 2. Fabricated by a combination of wet and
dry etching with metal liftoff, this design can
achieve the required spacings between the upper
and lower electrodes at their edges. Figure 3
shows various aspects of completed genosensor
devices.
for DNA Decoding
Sponsor
1.2.2 Genosensor Fabrication Development
Houston Advanced Research Center
Contract HRC-HG00665-01
The primary emphasis of this project is on the
process development and feasibility exploration for
the genosensor design of figure 2. For this chip,
conventional 2-pm photolithography, wet and dry
etching techniques, and electron-beam evaporation
and liftoff processes are used to fabricate an
interdigitated electrode structure in which the
opposing electrodes are separated vertically.
Project Staff
Dr. Daniel J. Ehrlich, Dr. Mark A. Hollis, Dr. Dennis
D. Rathman
During this past year, significant progress has been
made in process development.
Yield and
reproducibility of 6x6 passive arrays of various
325
Chapter 1. Genosensor Technology Development
GENOSENSOR SYSTEM
"AGTCG"
DNA PROBES WITH
HYBRIDIZED UNKNOWN DNA
Figure 1. Conceptual genosensor system.
developed techniques to connect Al wires directly to
Pt electrodes, eliminating the need for evaporation
and liftoff of Au bonding pads.
DETECTION-ELEMENT CROSS SECTION
Ti
A
\11.
J
AQUEOUS NA
SOLUTION
UNHYBRIDIZED
DNAPROBES
Si34
SiOn
SI SUBSTRATE
Figure 2. Electrode design for a permittivity genosensor.
The unit cell shown is repeated many times across a test
well to form an interdigitated test structure with the top
Au electrodes connected to one access line and the
bottom Au electrodes to the other.
active-area sizes have been dramatically improved
to greater than 90 percent for all array sizes. Problems described in last year's report related to the
patterning of the narrow Pt interconnect lines have
been solved. In addition, the use of thicker Ti
layers underneath the Pt lines has resulted in better
adhesion of both the interconnects and the activearea electrodes to the substrates. We have also
326
RLE Progress Report Number 137
One of the more challenging fabrication efforts successfully completed this year was the development
of a buried-interconnect, or insulated-lead, process.
(The motivation for developing this process is discussed in Section 1.2.3 below). This involves the
use of photosensitive polyimide as a second-layer
dielectric which is deposited on the completed
The active electrode area is opened
device.
conventional
by
polyimide
the
through
photolithography, where the polyimide behaves like
positive photoresist which can be developed out
after exposure. The polyimide is then hard baked
at 2000C or greater and remains on the genosensor
chip to insulate the interconnect leads from the
chemical solutions used in the attachment and
measurement of DNA. In initial tests, the polyimide
does seem to be somewhat degraded by the chemical treatments required for DNA strand attachment,
but not so severely that the electrical measureInvestigation of these
ments are compromised.
matters is continuing.
Chapter 1. Genosensor Technology Development
Figure 3. Collection of photographs showing various views of a complete genosensor device. (a) Top view of the
standard 100 pm X 100 pm single test cell. (b) Top view of the 6 X 6 passive array of test cells. (c) Scanning electron
micrograph (SEM) closeup of the interdigitated electrode fingers. (d) Genosensor chip in electronic package.
During this past year, we have delivered 30 packaged devices (and several unpackaged chips for
DNA attachment experiments) to HARC for tests for
this project. At present, several additional wafers
are in various stages of processing.
1.2.3 Genosensor Test Results
Extensive experimental and theoretical studies of
genosensor electrical properties have been carried
out this past year. It has been found that the preliminary data showing peaks in dissipation factor for
DNA probes and hybridized DNA that were shown
in figure 4 in last year's report are actually artifacts
of parasitic effects. The first-generation genosensor
used for those measurements did not have insulated interconnect leads on the chip surface, thus
the leads were exposed to the aqueous saline solution used during the electrical tests. Extensive
electromagnetic modeling has shown that the par-
ticular parasitic resistance and capacitance associated with the leads combined with the high
dielectric constant and conductivity of the saline can
reduce the phase velocity of electromagnetic propagation along the leads by as much as a factor of
100. This slow-wave effect produces an anomalous
inductive behavior in the measurement circuit,
causing spurious resonances which show up as the
peaks in last year's data. These findings are the
motivation behind the development of the insulatedlead process described above.
It should be
stressed that this effect is only important in interconnect leads that run across the chip surface from
the bonding pads to the sensor sites and is not
important in the interdigitated electrodes in the
sensor sites themselves.
Measurements on the new devices having insulated
leads are being performed presently. It appears
that the permittivity of DNA in aqueous solution can
be sensed, especially below 300 kHz. Detailed test
327
Chapter 1. Genosensor Technology Development
S10
SUBMICRON
PERIOD
2
INSULAT
DNA
POLYSILICON
ELECTRODE
(GOOD DNA FILL FACTOR)
Figure 4. Advanced genosensor design with high-aspect-ratio insulated electrodes.
results will be reported next year after the data
have been extensively analyzed.
1.3 Microdetection Technology for
Automated DNA Sequencing
Sponsor
Houston Advanced Research Center
Contract HRC-HG00776-01
Project Staff
Dr. Daniel J. Ehrlich, Dr. Mark A. Hollis, Dr. Dennis
D. Rathman
The primary emphasis of this effort is to complement the program described above by optimizing
the electrode geometry for maximum sensitivity.
Initial work for this contract began with the
electrode configuration illustrated in figures 2 and 3.
Subsequently, both experimental studies and theoretical modeling have shown a need to design and
fabricate more aggressive electrode geometries,
which are now being developed as described
below.
Measurements by our colleagues at HARC and
Baylor College of Medicine have shown that DNA
attachment to SiO 2 is more easily achieved than
attachment to Pt or Au. In fact, surface-attachment
densities of DNA probes are typically one order of
328
FLE Progress Report Number 137
magnitude higher on oxide-coated polysilicon compared to the Pt electrodes of our conventional permittivity chip designs. The exact reasons for this
are not yet known but may be related to the different linker chemistries used for attachment or to
the strong ionic and polarization effects observed at
liquid-metal interfaces. In either case, the fabrication of a permittivity chip incorporating oxidecoated polysilicon electrodes is currently underway.
Because sophisticated dry-etching technology is
available for patterning polysilicon, electrode structures having large height-to-spacing ratios should
Several wafers are currently in
be possible.
process which have coplanar, nonself-aligned
oxide-insulated polysilicon electrodes. This design
is conceptually illustrated in figure 4.
In addition to this coplanar design, we are also fabricating one using the self-aligned electrode architecture illustrated in figure 2 but having
submicrometer-periodicity oxide-insulated polysilicon electrodes. The patterning of the electrodes is
being done by a combination of laser interferonletric
For the
lithography and reactive ion etching.
attachment chemistry that will be used with these
devices, the DNA probes will probably attach not
only to the electrodes but also to the oxide/nitride
This will substantially
sidewalls of the wells.
increase the probe density in each sensor site and
possibly the detection sensitivity. Several wafers in
which we use this approach are currently in process
as well.