Note: Descriptions are shown in the official language in which they were submitted.
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DEVICE FOR DETECTING BIOLOGICAL AND CHEMICAL PARTICLES
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. ~119(e) to U.S.
Provisional Application No. 60/43,274, filed June 27, 2003, and to U.S.
Provisional
Application No. 60/551,131, filed March 8, 2004, the complete disclosures of
which
are expressly incorporated by reference herein.
FIELD OF THE INVENTION
This disclosure relates to devices, methods of use, and methods of
fabrication of such devices, for use in selectively concentrating, capturing,
and
detecting the presence of species, for example, chemical molecules such as
gases or
proteins, biological species, such as cells (e.g., bacterial cells and/or
eukaryotic cells)
or virus particles, spores, molds, yeast, microorganisms, and the like. The
invention
is disclosed in the context of arrays of micro- and nano-scale structures
intended to be
used, for example, as mass sensors to detect the presence and/or
concentrations) of
the selected species. However, it is believed to be useful in other
applications as well.
BACKGROUND
Detection of very small quantities of chemical and biological species,
for example, virus particles, bacteria, chemical molecules, or any other
analyte, is
important in industrial, environmental, human health, and bio-security
applications, to
name a few. Various methods, devices, and structures for the detection of
chemical
and biological species utilize a variety of technologies and methods. However,
many
prior methods, devices, and structures lack the ability to detect low
concentrations of
such species, and require long turnaround times. Many such methods and
apparatus
also generally require chemical or biological labeling of the species to be
detected.
All of these characteristics can complicate the detection process.
Currently, a few reliable detection methods for some minute analytes
do exist. For example, in the field of viral detection, methods for detection
of
coronaviruses include virus isolation, electron microscopy, and
immunofluresence
assay with human coronavirus specific fluorescein (hereinafter sometimes FITC)-
conjugated antibodies. Additionally, some molecular detection methods utilize
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polymerise chain reaction (hereinafter sometimes PCR) amplification for
screening
for viruses such as coronaviruses. However, for the detection of many viruses,
including coronavirus, the above methods typically require dedicated
equipment,
facilities, and skilled persomiel to carry out the tests.
Viruses are traditionally quantified by their replication-induced
cytopathic effects on cultured cells or by immunological means such as ELISA
or
immunoelectron microscopy. The cell culture quantification methods usually
take
several days to weeks and some viruses such as some human respiratory
coronaviruses cannot be grown in cultured cells ifi. vitYO. The ELISA method
involves
the capture of viruses by an antibody cross-linked to a surface, such as 96-
well
polystyrene microtiter plates. The captured viruses are then detected with a
secondary
antibody that is attached to an enzyme such as horseradish peroxidase, wluch
catalyzes a chemical reaction that produces a color change. Cell culture,
ELISA and
immunoelectron microscopy techniques typically are labor-intensive techniques
that
require experienced personnel to perform.
Real-time quantitative PCR amplification assays have recently come
into extensive use to identify and quantify pathogenic RNA and DNA viruses,
such
HIV-1, HIV-2, hepatitis B and C viruses, dengue virus, and cytomegalovirus
(see, for
example, Mackay, I. M., Arden, K. E., Nitsche, A., Real-time PCR in Virol.,
Nucleic
Acids Res., 15: 1292-1305, 2002, incorporated herein by reference). These
assays are
highly sensitive and faster than prior methods. However, due to the extreme
sensitivity of the method, PCR-based assays are troublingly prone to
contamination,
which can result in false positive detection. Moreover, PCR-based assays
generally
are not suitable for high throughput detection and discrimination of viral
subtypes or
~5 antigenic variants.
Micro- and nano-scale technology has been increasingly used in a wide
variety of chemical and biological applications, including, for example,
detection and
characterization of biological species in biomedical applications. For
example, one
area of technology that has become increasingly important but not very well
developed is the handling, manipulation, and characterization of viral
infectious
agents, for example, the detection of very low virus particle concentrations
in air and
their continuous monitoring from air, using biomedical micro-electromechanical-
systems technology (BioMEMS). In addition, reducing the time-to-result to be
able
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to perform 'point-of use' analysis has become very important. Micro- and nano-
scale
devices could not only yield very important scientific data but could also be
used in
practical diagnostic applications in the health and food industries, and in
biological
and chemical hazard prevention systems.
Cantilevers, bridges, or other micro- and nano-scale suspended
structures associated with semiconductor devices have been used to selectively
detect
biological and chemical particles, for example, as sensors or actuators in
atomic force
microscopy (AMF) and scanning probe microscopy (SPM). When the mass of the
particle to be detected is added to the suspended structure, surface stresses,
vibrational
deflections, and like parameters of the structure are detectably altered, for
example,
by changing the resonant frequency of the structure. It has recently been
demonstrated that biomolecular interactions such as DNA-DNA and protein-ligand
interaction on cantilever surfaces cause bending of microcantilever beams
(see, for
example, Raiteri, R., Nelles, G., Butt, H.-J., Knoll, W. and Slcladal, P.,
Sensing of
biological substances based on the bending of microfabricated cantilever.
Sensors
Act. B. 61: 213-217, 1999; Fritz, J., Bailer, M. K., Lang, H. P., Rothuizen,
H.,
Vettiger, P., Meyer, E., Guntherodt, H., Gerber, C., Gimzewski, J. K.,
Translating
biomolecular recognition into nanomechanics, Science, 288: 316-318, 2001, all
of
which are incorporated herein by reference). The method was found to be
sufficiently
sensitive that it could differentiate single base-pair mismatches in a DNA
hybridization event (see for example, Hansen, K. M., Ji, H. F., Wu, G., Datar,
R.,
Cote, R., Majumdar, A., Thundat, T., Cantilever-based optical deflection assay
for
discrimination of DNA single nucleotide mismatches, Anal. Chem., 73: 1567-
1571,
2001, incorporated herein by reference).
Two primary methods for detecting the vibrational deflection of
structures such as cantilevers and bridges are optical lever detection and
piezo-
resistive detection. Optical lever detection techniques are used, for example,
to
measure the deflection of atomic force microscopy (AFM) tips. A laser beam is
reflected off of the tip of a cantilever and is typically detected by two
photodetectors.
The relative intensity of the laser signal received by the two photodetectors
is related
to the degree to which the cantilever is deflected. However, this technique
has several
shortcomings, especially as the dimensions of cantilevers are reduced in order
to
detect particles having sub-100 femtogram (fg) masses. First, the technique
requires
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reflecting a focused laser beam off the surface of the cantilever tip,
resulting in larger
lateral dimensions of the cantilever. Second, the technique generally
precludes
simultaneous monitoring of an array of cantilevers, which is necessary or
highly
desirable in a variety of detection applications. Finally, a relatively large
off chip
system is required, including the laser, focusing optics, and photo detectors.
Therefore, the degree to which such a system can be miniaturized is limited.
Piezo-resistive detection methods eliminate the requirement of external
optical system elements by electrically measuring the deflection-induced
strain on the
surface of the cantilever or the change in vibration frequency due to the
particle mass.
The technique is analogous to macroscale strain gauges that are attached to
mechanical components to measure deformation. The change in resistance for a
piezo-electric element in response to strain is induced by cantilever
deflection. This
change in resistance can be detected, for example, by using a Wheatstone
bridge
circuit. Piezo-resistor elements can be built into the surface of the
cantilever by
standard integrated circuit fabrication techniques. This provides a flexible,
integrated,
and scaleable vibration sensing technique adaptable to a variety of cantilever
structures.
When a cantilever is deflected, a strain gradient is induced throughout
the body of the cantilever, with zero strain at the center. One surface is in
tension
(positive strain) while the opposite surface is in compression (negative
strain). Since
the response of a piezo-resistive element to strain is approximately symmetric
about
zero strain, a piezo-resistor that penetrates through the entire cantilever
would not
respond to the flexion. The response of the top half of the element would be
equal
and opposite to that of the bottom half, resulting in no net response.
Therefore, to be
effective, piezo-resistive elements are generally placed near the surface
region of the
cantilever. While this may be accomplished by implantation and/or diffusion
into
relatively thick cantilevers, it becomes very difficult to achieve in sub-100
nanometer
(mn) thick cantilevers which are generally required for detecting sub-100 fg
masses.
Accordingly, devices, methods of use, and methods of fabrication of
such devices, for use in selectively concentrating, capturing, and detecting
the
presence of species, for example, chemical molecules such as gases or
proteins,
biological species, such as cells (e.g., bacterial cells and/or eukaryotic
cells) or virus
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particles, and the like are desirable.
SUMMARY
According to one aspect of the invention, an apparatus comprises a
filter structure adapted to be coupled to an alternating current voltage
source. The
filter structure includes at least one electrode having a feature which
promotes
establishment of at least one of a region of increased electric flux and a
region of
decreased electric flux relative to an adjacent region to select a first
species
susceptible of dielectrophoretic selection. The apparatus further includes a
cantilever
structure upon which the first species is collected. The apparatus also
includes at least
one device for causing the cantilever structure to oscillate, and for
receiving as an
output from the cantilever structure the cantilever structure's frequency of
oscillation.
The at least one device is adapted to be coupled to means for determining from
the
output the mass of the first species collected on the cantilever structure.
According to another aspect of the invention, an integrated micro-
electromechanical analyte detection device comprises a substrate, a support
member
coupled with said substrate, a cantilever having a fixed end coupled to said
support
member and a free end, and a first electrode coupled to said substrate and
positioned
adjacent said free end of said cantilever such that the free end and the
electrode are
excitable terminals for dielectrophoresis.
According to another aspect of the invention, an integrated micro-
electromechanical analyte detection device comprises a substrate, a support
member
coupled to said substrate, a cantilever having a fixed end coupled to said
support
member and a free end, and a traalsistor having at least one region formed on
at least
one of said free end of said cantilever and a portion of said support member.
According to another aspect of the invention, an integrated micro-
electromechanical analyte detection device comprises a substrate, a support
member
coupled to or defined by said substrate, and a suspended member having first
and
second ends. At least the first end is coupled to the support member such that
at least
a portion of the suspended member is movable relative to the substrate. The
integrated micro-electromechanical analyte detection device further comprises
a field
effect transistor having a channel defined by said suspended member.
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According to another aspect of the invention, an integrated micro-
electromechanical detection device comprises a substrate, a first suspended
member
having first and second ends, at least said first end fixed with said
substrate, a
dielectrophoresis filter coupled to said substrate, and a fluid channel
defined by said
substrate and communicating fluid between said first suspended member and said
dielectrophoresis filter.
According to another aspect of the invention, a method is provided for
determining a characteristic of an analyte interacting with an integrated
detection
device. The device includes a substrate and a suspended member having first
and
second ends. At least the first end is coupled with the substrate. The method
comprises applying an electric signal to the suspended member to perform
dielectrophoresis on the analyte and determining a change in a parameter of
the
suspended member.
According to another aspect of the invention, a method is provided for
determining a characteristic of an analyte interacting with an integrated
detection
device. The integrated detection device includes a substrate, an electrode and
a
suspended member having first and second ends. At least the first end is
coupled with
the substrate. The method comprises applying an electric signal to the
electrode and
the suspended member to perform dielectrophoresis on the analyte.
According to another aspect of the invention, a method is provided for
determining a characteristic of an analyte interacting with an integrated
micro-
electromechanical detection device. The device includes a substrate, a support
member coupled with the substrate and a suspended member having first and
second
ends. At least the first end is coupled to the support member. The method
comprises
determining a change in a current signal conducted by a transistor region
defined at
least in part by one of the suspended member and the junction of the first end
of the
suspended member and the support member, upon the analyte interacting with the
suspended member.
According to another aspect of the invention, a method is provided for
fabricating an integrated circuit on a substrate. The integrated circuit
includes a
cantilever. The method comprises providing a silicon-on-insulator (S01) wafer
having a buried oxide layer (BOX), thinning the SOI layer to less than
approximately
30 nm, photolithographically patterning and etching the SOI layer to define
the
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cantilever, thinning the exposed portions of the BOX layer, depositing an etch
stop
layer to the exposed portions of the SOI and BOX layers, photolithographically
patterning an etch window extending laterally approximately from the free ends
of the
cantilever and vertically to at least the depth of the base of the SOI layer,
and etching
the BOX layer below the cantilever, thereby releasing an end of the
cantilever.
According to another aspect of the invention, a method is provided for
fabricating an integrated circuit on a substrate. The integrated circuit
includes a
cantilever. The method comprises patterning a seed window using an insulator
mask
on substrate, providing an oxide layer displaced vertically above the seed
window and
laterally overlying the seed window and at least a portion of the insulator
mask,
growing silicon selectively from the base of said seed window vertically to
the top of
the insulator mask and laterally between the insulator mask and the oxide
layer, and
etching at least a portion of the oxide layer and the insulator mask, thereby
releasing a
suspended end of the selectively grown silicon from the remaining structure.
According to another aspect of the invention, a method is provided for
fabricating an integrated circuit on a substrate. The integrated circuit
includes a
cantilever and a field effect transistor. The method comprises providing a
silicon-on-
insulator (SOI) wafer, thinning the SOI in the area where the cantilever will
be
formed using an anisotropic etch in order to form an abrupt step adj acent the
top
surface of cantilever and the junction of the remaining structure, oxidizing
the silicon
surface of the cantilever and the step to form transistor gate oxide,
depositing
polysilicon gate material confonnally along the top surface of the cantilever
adjacent
the step, anisotropically etching the polysilicon gate material through the
entire
deposited thiclcness so that a sidewall of polysilicon having a lateral width
approximately equal to the deposited thiclmess remains on any exposed vertical
surface, implanting a source and drain region using the polysilicon gate as an
implantation mask, and etching to release a suspending portion of the
cantilever on
the end opposite the transistor gate.
According to another aspect of the invention, a method is provided for
fabricating an integrated circuit on a substrate. The integrated circuit
includes a field
effect transistor (FET). The method comprises providing a silicon wafer,
thermally
growing a first thick oxide layer for substrate isolation, lithographically
patterning
and depositing a sacrificial silicon layer on the oxide layer, depositing a
second thick
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oxide layer using plasma enhanced chemical vapor deposition (PECVD), etching
via
holes to define the FET source and drain regions, and etching to selectively
remove
the sacrificial silicon layer. The removal defines a bridge formed by a
portion of the
second oxide layer suspended over the thermally grown oxide layer. The method
further comprises collapsing the oxide bridge to leave nano-scale via holes
near the
anchors of the oxide bridge, and wet etching a seed hole in the first oxide
layer in the
source region via hole. The seed hole extends down to the silicon layer
surface. The
method further comprises growing silicon epitaxially through the via holes,
removing
silicon remaining on the surface by chemical mechanical polishing (CMP),
implanting
the source and drain regions, and etching to remove the oxide encapsulating
the
epitaxially grown silicon. The silicon forms suspended nano-wires forming the
FET
channel and a thin connecting silicon plate between the wires. The method
further
comprises etching to remove the remaiiung oxide covering on the device and the
thin
silicon plate between the wires.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates schematically an exemplary integrated micro-
electromechanical analyte detection device constructed according to the
present
invention;
Fig. 2 illustrates a scanning electron micrograph of an exemplary
cantilever beam integrated into a semiconductor chip according to the present
invention;
Fig. 3 illustrates frequency shift of the cantilever illustrated in Fig. 2
after the addition of a single virus particle;
Fig. 4 illustrates a perspective view of an exemplary cantilever beam
and a supporting member in dynamic mechanical vibration;
Fig. 5 illustrates an exemplary cantilever beam and supporting member
in static deflection;
Fig. 6 illustrates graphically a desired resonant frequency and
minimum detectable mass for exemplary cantilevers having widths of l~.m and
various thicknesses;
Figs. 7 A-E illustrate cross-sectional views of an exemplary device in
various stages of semiconductor fabrication;
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Figs. 8A-B illustrate top views corresponding with the device of Fig.
7A-E in various states of fabrication;
Fig. 9 illustrates a system including scanning laser doppler vibrometer
determining the resonant frequencies of unloaded and loaded suspended members;
Fig. 10 illustrates graphically the resonant frequency shift measured
before various loaded cantilever beams measured by the system of Fig. 9;
Figs. 11 and 12 illustrate an exemplary method of fabricating an
ultrathin cantilever;
Fig. 13 illustrates a perspective view of an exemplary system for
measuring the unloaded resonant frequency of an exemplary cantilever using a
laser
and a detector;
Fig. 14 graphically illustrates the frequency response for the unloaded
cantilever illustrated in Fig. 13;
Fig. 15 illustrates the measurement of a loaded cantilever illustrated in
Fig. 13;
Fig. 16 illustrates the resonant frequency shift between the cantilevers
illustrated in Figs. 13 and 15;
Figs. 17A-D illustrate mechanical characterization of an exemplary
cantilever using known masses positioned at the free end;
Figs. 18 and 19 illustrate the resonant frequency change after the
additional of known masses for the exemplary cantilever illustrated in Figs.
17A-D;
Figs. 20-22 illustrate scanning electron micrographs of exemplary
embodiments of cantilevers;
Fig. 23 illustrates a cross-sectional view of the components and layers
of an exemplary cantilever having a sensing element at the junction of the
cantilever
and the support member according to the present invention;
Fig. 24 illustrates an elevation view of the areas of maximwn strain for
an exemplary cantilever according to the present invention;
Figs. 25A-D illustrate exemplary cantilever structures within fluid
channels defined in integrated semiconductor devices;
Figs. 26A-B illustrate a cross-sectional view axed a top view,
respectively, of an exemplary cantilever having a transistor region adj acent
the area of
maximum strain of the cantilever;
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Fig. 27 graphically illustrates the carrier density for the transistor
region of Figs. 26A-B for various depths from the surface of the cantilever;
Fig. 28 illustrates DEP motion of particles between an electrode and a
plate;
Fig. 29 illustrates the use of DEP to concentrate analytes at free ends
of a cantilever pair according to the present invention;
Fig. 30 illustrates a perspective view of an array of cantilever pairs
using DEP to concentrate analytes at the free ends of the cantilevers;
Fig. 31 illustrates binding of LiSteria moyaocytogehes on various
surfaces;
Fig. 32 illustrates a perspective view of a cantilever having binding
agents for a target analyte and using mechanical vibration of the cantilever
to aid in
the release of non-target species;
Fig. 33 illustrates individual elements for selectively binding a target
analyte to an exemplary cantilever;
Fig. 34 illustrates a schematic diagram of a DEP filter for a fluid
channel for a device such as that illustrated in Fig. 1;
Fig. 35 illustrates a scanning electron micrograph of fluid channels and
electrodes for an exemplary DEP filter such as for the exemplary embodiment
illustrated in Fig. 1;
Figs. 36A-C illustrate DEP manipulation and separation of live and
dead cells on electrodes;
Fig. 37A-B illustrate an exemplary DEP filter for selectively capturing
a target analyte;
Fig. 38 illustrates an optical image of a device used for the DEP
manipulation illustrated in Figs. 36A-C;
Figs. 39A-B illustrate scanning electron micrographs of a device
having suspended nano-wire members;
Figs. 40A-G illustrate perspective views of a process for fabrication of
the devices illustrated in Figs. 39A-B;
Figs. 41 B, C, E and G are detailed views more fully illustrating the
fabrication steps of Figs. 40 B, C, E and G, respectively;
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Fig. 42 illustrates the change in conductance of an exemplary
suspended member after cycling between exposure to oxygen and purging in
nitrogen;
Fig. 43 graphically illustrates the cycling illustrated in Fig. 42 with the
addition of a heat cycle;
Fig. 44 illustrates a schematic elevational view of the structure of an
integrated detection device having a suspended channel;
Fig. 45 illustrates a perspective view of an exemplary integrated device
having suspended nano-wire members connected by an ultra thin silicone film
according to the present invention;
Fig. 46 illustrates the exemplary device of Fig. 45 after the removal of
the silicon film connecting the suspended nano-wires; and
Fig. 47 illustrates a perspective view of an exemplary device having a
suspended member with an electrode.
Corresponding reference characters indicate corresponding parts
throughout the several views. The exemplification set out herein illustrates
an
embodiment of the apparatus and such exemplification is not to be construed as
limiting the scope of this application in any manner.
DETAILED DESCRIPTIONS OF ILLUSTRATIVE EMBODIMENTS
An exemplary micro- or nano-electromechanical analyte detection
device includes a cantilever, nano-wire, or other suspended member for
detecting
selected biological or chemical species based on the change in resonant
frequency,
surface stress, or other characteristic of the suspended member upon
interaction with a
selected analyte. For the purpose of this disclosure, analyte is defined as
any
biological or chemical species, including, for example, chemical molecules,
proteins,
bacteria, cells, virus, spores, molds, yeast, microorganisms, and the lilee.
DEP can be
utilized to concentrate a selected analyte, for example at the free end of the
cantilever
or other suspended member. The suspended member may also include a region of a
transistor, for example a channel of a field affect transistor to provide on-
chip
electronic detection of selected analytes. Integrated devices having arrays of
suspended members for detecting selected analytes located within a fluid
channel are
also disclosed as well as methods of use and methods of fabrication of the
exemplary
integrated devices.
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The recent technological advances in nanotechnology and
micromachinirig of semi-conductor materials present new opportunities for
inexpensive, small and sensitive diagnostic or other detection devices capable
of rapid
and highly accurate detection of minute analytes, for example, sub-100 fg
analytes.
This disclosure relates to a device that can detect the presence of analytes
by either
changes in surface stress or detection of added mass through a change in
resonant
frequency of a suspended structure, for example, a cantilever or bridge
structure.
One exemplary embodiment of the present device includes a micro-
cantilever-based virus detection device and technique which may yield
performance
characteristics exceeding the sensitivity and specificity of present detection
techniques, for example, PCR amplification assays and ELISAs. Calculated
limits of
detection of the exemplary device are 10-17-10-1$ grams (gm) of mass change on
the
cantilever surface. This translates to the mass of single virus particles.
When this
method is coupled to currently available monoclonal antibodies against viruses
(see,
for example, Parren, P. W., and Burton, D. R., The antiviral activity of
antibodies ih
vitro and Z32 v11~0, Adv. Tm_m__unol., 77, 195-262, 2001, incorporated herein
by
reference), its specificity could surpass ELISAs since the described technique
does
not rely on enzymatic reaction kinetics as do the mentioned prior art
techniques. The
ability to detect and monitor in real-time and on a continual basis viruses
and their
subtypes, particularly the most contagious viruses and bioterrorism agents,
can have
dramatic implications in the confinement and management of the viral
epidemics.
One of the most contagious forms of disease spread occurs via
aerosolized pathogens. Recently, considerable attention has been given the
deadliest
bioterrorism agents that spread through airborne particles, such as smallpox
virus and
anthrax toxin. Although influenza viruses typically are not considered
bioterrorism
agents, these viruses can be pandemic with devastating casualties. Human
rhinoviruses and coronaviruses are among the most common causes of upper-
respiratory infectious diseases, and clearly, a device to rapidly measure
these agents
in air samples can have profound practical and economic implications.
Thus, there is an important need for a micro-scale, robust, real-time
monitoring device, based on integrated micro-machined ultrathin cantilever
arrays
with on-board signal processing for the rapid and sensitive detection of
infectious
agents in field settings and in primary patient care facilities. According to
one
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exemplary embodiment, an array is specific for specific pathogens and has the
sensitivity to detect a single virus or toxin molecule. The exemplary
embodiment
includes a dielectrophoresis (hereinafter sometimes DEP)-based infectious
agent
trapping, separation and concentration device for the detection of an airborne
virus on
functionalized micro-scale cantilevers.
The exemplary device provides all-electronic detection of single
entities in the zeptogram andlor attogram range, has a short turnaround time
of
detection, and does not require labeling of entities. The device includes an
ultrathin
and highly sensitive mechanical structure built on an electronic chip that
detects
DNA, proteins, viruses, and other analytes in very low concentrations. The
device
may also detect the composition of analytes. By tailoring the function of the
mechanical device surface, specific adsorption of biological or chemical
species can
be achieved, thus permitting target-specific detection.
The exemplary device includes nano-mechanical suspended structures,
for example, cantilever ("diving board") or bridge (for example, nano-wire)
sensors.
An exemplary method of device fabrication produces ultra-thin, and thus,
highly
sensitive suspended structures (less then 500 nrn thick, 130 ~m long, and 20
~.m wide;
preferably less than 30 rim thick, 5 ~m long, and 2 ~,m wide; more preferable
less
than 10 nm thick, 3 ~,m long, and 1 ~,m wide; more preferably approximately
100
angstroms (~) thick) on a silicon or other semiconductor chip. The exemplary
chip
provides detection of analytes, for example, DNA, proteins, viruses, and
chemicals, in
very low concentrations, for example, less than 10 particles per milliliter.
These
suspended structures can detect the presence or binding of analytes on them,
for
example, by a resulting change in resonant frequency due to the added mass or
a
resulting change in surface stress due to the change in surface energy. While
the
suspended member of some exemplary embodiments comprise silicon, other
semiconductor, metallic conductor, or non-conductive materials may be
alternatively
used.
The suspended structures, for example cantilevers, can be arrayed
within a micro-fluidic channel where an air or other fluid flow can be passed
over the
structures. When an alternating current (ac) DEP signal (with frequency
different
than the resonant frequency) is applied to electrodes associated with the
suspended
structures, particles having permittivities greater than the carrier fluid
(gas or liquid)
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will be trapped near the regions of highest field gradient, for example, at
the tips of
the suspended structures. If the suspended structures are coated with
antibodies for
specific viruses, only those viruses are captured. Any non-specifically bound
particles can be removed by driving the suspended structures in resonance, at
an
appropriate amplitude, while the specifically bound particles remain attached
to the
antibodies present on the suspended structures. Then the attached mass can be
detected by measuring a shift in the resonance frequency of the suspended
structures
due to the attached mass.
Mechanical vibrations or bending of suspended structures are typically
detected by a laser reflection based system, which is not amenable to
miniaturization.
Capacitive detection methods may not be practical for low capacitance
applications
due to the signal levels, and hence susceptibility to noise. In order to
obtain useful
signal levels, the total capacitance is sometimes increased by increasing the
overlap
area of the capacitive regions, and by keeping a minimum distance between the
sensing electrodes. However, for accurate mass detection in the attogram
range, the
resonating structure should be as small as possible, thus limiting the
available signal
for capacitive detection.
In one exemplary embodiment of the novel resonating channel
structure, the electrode used to drive the device may be the same electrode
that
supplies an electric field for the device. In an exemplary embodiment wherein
the
device includes a field effect transistor (FET), the electrode is the gate or
the channel
of the transistor. For example, an ultratlun suspended silicon bridge, for
example, a
nano-wire, provides the FET channel and an active resonant sensing area. The
suspended structure may be grown, for example, by tunnel epitaxial growth,
where
the growth path of the silicon is restricted and is allowed only in one
direction in a
cavity. During the formation of the cavity, an oxide layer forming a roof of
the cavity
can be made to collapse due to stiction and subsequent growth of silicon
results in
very thin single crystal silicon wires suspended between two islands or
supporting
structures, which may form the source and drain of the FET.
Alternatively, by not doping the source and drain regions, the nano-
wires can be simply used as resistors. Resistive nano-wires may provide
detection of
molecules binding on the wire by measuring resistance changes across the nano-
wire.
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By oscillating the nano-wire channel using the FET gate, the current
through the FET is modulated as a function of the distance between the
suspended
channel and the gate. Also, the current will be a function of the gate bias,
as in typical
FETs. The piezo-resistive effects on oscillating nano-wire chamzels also
affect the
modulation of current due to stress changes that the channel experiences due
to
mechanical oscillation. By monitoring the drain-source current through the
FET, the
amplitude and frequency of the oscillations can be determined, with high
signal levels
and high signal-to-noise ratios compared to typical capacitive sensing. Thus,
an
advantage to having the nano-wire or other suspended structure comprise the
FET
channel, rather than the FET gate, is the combined effect on the channel
current of
both the physical displacement of the channel relative to the gate and the
piezo-
resistive change from the bending of the channel. Whereas, if the nano-wire
comprises the FET gate, the piezo-resisitive effect acts on the gate while the
displacement effect acts on the channel.
Because the oscillation frequency and amplitude can be sensed
electically, and high signal-to-noise ratios are expected, very small changes
in
resonant frequncy, due to the added mass of particles on the nano-wires can be
resolved by incorporating feedback. For a single crystal silicon (for example,
E .=162
GPa and density ~2.3 3g/cm3) nano-wire beam, having a length of 5 pm , width
of 3
Vim, and thickness of O.Ol p,m, the calculated resonant frequency is about
2.123340
MHz. Assuming a minimum detectable frequency of 1 Hz, the miiumum detectable
mass is estimated to be, 5.5 X 10-198, which corresponds to the approximate
mass of a
single protein, therefore smaller than the mass of a single virus particle or
cell.
Alternatively, the exemplary device can be operated in other modes of
electrical operation and/or mass detection, for example, as resistors,
transistors, or
capacitors with detection of a change in surface potential or a change in
mass.
Referring now to Fig. 1, one exemplary embodiment of the device may
be an integrated micro-electromechanical analyte detection device 100
comprising
integrated circuit 102 which has been fabricated to include all device
components, for
example, fluid channel 104, cantilever arrays 106, 108, and 110, DEP filters,
concentrators, or sorters (which may be configured to filter, concentrate, or
sort
particles; however, herein sometimes collectively referred to as "filters")
112, 114,
and 116, signal processing and control circuitry 118, and fluid intake filters
120 and
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exhaust filters 122. Integrated device 100 is capable of concentrating,
capturing, and
detecting the presence of selected chemical and/or biological analytes.
Cantilevers 106, 108, and 110, or other suspended structure, for
example, a bridge, has small enough dimensions to detect analytes having sub-
100 fg
masses, for example, individual bacterial cells, viruses, or chemical
molecules.
Integrated device 100 may include structures and circuitry 118 for measuring
changes
in surface stress, energy, deflection, or resonant frequency of cantilevers
106-110
induced by the added mass or other characteristic of an analyte bound or
otherwise
interacting with cantilevers 106-110. For example, referring to Figs. 2 and 3,
the
resonant mechanical oscillation (or vibration) frequency of cantilever 106,
which is
shown in Fig. 2 and is supported by support member 130, is decreased by a
measurable quantity by the resting of analyte 132 on cantilever 106. For the
example
shown in Fig. 2, analyte 132 is a Vaccinia virus particle. Referring to Fig.
3,
unloaded cantilever beam oscillation 134 has a peak resonant frequency of 1.27
megahertz (MHz) and loaded cantilever beam oscillation 136 has a peak resonant
frequency of 1.21 MHz. As will be further discussed below, and as is generally
known in the art, the shift in resonant frequency is directly related to the
mass and
therefore the identity of analyte 132 interacting with cantilever 106.
Refernng again to Fig. l, individual or sets of cantilevers 106, 108,
and 110 may be functionalized in order to provide sensitivity to particular
biological
or chemical analytes. For example, cantilevers 106-110 may be coated with
different
antibodies, antigens, or other binding agents to promote binding of a
particular
analyte to the individual cantilever 106-110, or may be treated with an anti-
fouling
agent to prevent non-specific or undesirable specific particles from
interacting with
cantilevers 106-110. Additionally, DEP filters 112-116 may be utilized in
conjunction with cantilevers 106-110 in order to selectively sort, concentrate
or
capture specific analytes or other particles and direct them toward or away
from
cantilevers 106-110.
Known nanomechanical sensors can detect single individual
biochemical molecules (see for example, M. L. Roukes, Sci. Am. 285, 48
(September
2001, incorporated herein by reference). Operating as resonance detector based
mass
sensors, known microstructures can detect individual bacterial cells (see, for
example,
Ilic, et al., 2001). For cantilever beam 140, shown in Fig. 4, free end 142 is
located
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opposite fixed end 144, which is mechanically coupled to support member 146.
For
cantilever beam 140, the change in mass as a function of change of resonant
frequency. Assuming all the mass is added right at free end 142, is given as,
_ k 1 _ 1
4~ ~ .fit .f ~ ( )
0
where lc is the spring constant, fo is the unloaded resonant frequency
and fl is the loaded resonant frequency. As can be understood, the way to
improve
the mass sensitivity (that is, larger frequency shift for a smaller mass
loading) is to
decrease k as well as increase the resonant frequency. This can be done by
decreasing
the size of the cantilever beam 140 as well as decreasing tluckness 148 of the
cantilever beam.
Single-crystal materials are generally used to make sensor elements
due to their high mechanical quality factor. Silicon, for example, can be used
for
fabricating sensor elements such as cantilever 140, due to advantages such as
low
stress and controlled material quality, using currently available VLSI circuit
fabrication facilities, miniaturization of devices, high control of
dimensions, and the
economical advantage of batch fabrication. In addition, if piezo-resistive
detection
modes are preferred, for example, utilizing piezo-electric element 150 shown
in Fig.
5, especially due to the need for arrays of cantilevers and detectors, then
silicon
provides the capability to realize such elements 150 to detect deflections.
Cantilever beams were first introduced to the nanotechnology field
with their use as force sensors in atomic force microscopy (AFM) (see Binnig,
G.,
Quate, C. F. and Gerber,. Atomic force microscope, Physical Review Letters,
56:930-
933, 1986, incorporated herein by reference). They have also been used
extensively
as probes in various other imaging teclu~iques, involving different
interactions
between the probe and the sample, (see, for example, Wickramasinghe, H.K.
Progress in scanning probe microscopy , Acta Materialia, 48:347-358, 2000;
Moy, V.
T., Florin, E-.L. and Gaub~ H. E. Intermolecular forces and energies between
ligands
and receptors , Science, 266:257-259, Oct. 1994; Lee, G.U., Chrisey, L.A. and
Colton, R. J. Direct measurement of the forces between complementary strands
of
DNA , Science, 266:771-773, 1994; Dammer, U., Popescu, O., Wagner, P.,
Anselmetti, D., Guntherodt, H-.J. and Misevic, G. N. Binding strength between
cell
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adhesion proteoglycans measured by atomic force microscopy , Science, 267:1173-
1175, 1995; Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K.
and
Schindler, H. Detection and localization of individual antibody-antigen
recognition
events by atomic force microscopy , Proceedings of the National Academy of
Sciences of the United States of America, 93:3477-3481, 1996; Rief, M.,
Gautel, M.,
Oesterhelt, F., Fernandez, J. M., an Gaub, H. E. Reversible unfolding of
individual
thin irmnunoglobulin domains by AFM, Science, 276:1109-1112, 1997; Radmacher,
M. Fritz, H. G. Hansma, and P. K. Hansma, Direct observation of enzyme
activity
with the atomic force microscope , Science, 265:1577-1579, Sept. 1994;
Gimzewski,
J. K. and Gerber, C. A ferntojoule calorimeter using micromechanical sensors ,
Review Sci. Inst., 65:3793-3798, 1994; and Barnes, J. R., Stephenson, R. J.,
Woodbum, C. N., O'Shea, S. J., Wetland, M. E., Rayment, T. , Gimzewski, J. K.
and
Gerber, C. A femtojoule calorimeter using micromechanical sensors , Review
Sci.
hlst., 65:3793-3798, 1994; all of which are incorporated herein by reference).
Additionally, surface-stress change induced by deflection of cantilevers has
been
noted (see, for example, Butt, H.-J. A sensitive method to measure changes in
the
surface stress of solids , J. Colloid Interface Science, 180:251-260, 1996;
Fritz, J.,
Baller, M. K., Lang, H. P., Rothuizen, H., Vettiger, P., Meyer, E.,
Guntherodt, H.-J.,
Gerber, C. and Gimzewski, J. K. Translating biomolecular recognition into
nanomechanics , Science, 288:316-318, 2000; Berger, R., Delamarche, E., Lang,
H.
P., Gerber, C., Gimzewski, J. K., Meyer, E. and Guntherodt, H.-J. Surface
stress in
the self assembly of alkanethiols on gold , Science, 276:2021-2024, 1997; and
Wu,
G., Datar, R.H., Hansen, K.M., Thundat, T., Cote, R.J. and Majumdar, A.
Bioassay of
prostate-specific antigen (PSA) using microcantilevers , Nature Biotech.,
19:856-860,
2001; all of which are incorporated herein by reference), based on static
bending 152
of the cantilever beam 140, as shown in Fig. 5. Alternatively, cantilever beam
140
deflections 154 can be measured in the dynamic mode allowing cantilever beam
140
to be used as a micro-mechanical oscillator sensor. Additionally, static 152
or
dynamic 154 deflections of suspended structures may be measured by the change
in
capacitance or current flow associated with regions of the suspended
structures and
adjacent structures as will be further discussed below.
Fabrication of exemplary embodiments of the present device may
include selective epitaxial growth (SEG), epitaxial lateral overgrowth (ELO),
and
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chemical mechanical polishing (CMP) for the micro-fabrication of thin single-
crystal
silicon cantilever beams or other suspended structures. The thin suspended
structures
can be integrated into silicon-based micro-systems such as flow sensors,
pressure
sensors, bio-chemical sensors and the like. The disclosed fabrication process
can
produce low stress sub-100nrn thickness cantilevers 140 for ultra-high
sensitivity
chemical and biological detection.
The minimum detectable mass with a resonant cantilever beam will
depend on the geometry and the material that the cantilever is made of, as
well as the
minimum detectable frequency change of the cantilever beam. Also, the location
of
the mass to be detected on cantilever 140 will effect the sensitivity. If the
mass is
placed at free end 142 of cantilever 150, the device can detect lower masses,
since the
effective mass will be higher closer to free end 142 of cantilever 140.
The resonant frequency of cantilever 140 assuming small deflection on
free end 142 of cantilever 140 (the mechanical system can be modeled as a mass
and
spring for small deflections) can be expressed as:
_1 k (2)
.fo = 2TC mel!'
where meff is the effective mass of cantilever 140, and k is the spring
constant. The
spring constant will be dependent on the geometry of cantilever 140. For a
rectangular
cantilever with dimensions 1 as length 158, w as width 156, t as thickness
148, and a
material modulus of E, the spring constant can be expressed as,
k 4l W (3)
An effective mass is needed since the mass of cantilever 140 is not
concentrated at free end 142, but distributed. Effective mass can be expressed
in terms
of the cantilever mass by Tn~~. = 0.24 ' Yi2~arttilever ~ For a mass added to
free end 142 of
cantilever 140, the resonant frequency of the system will decrease. The new
resonant
frequency can be expressed as:
.~ _ _1 k (4)
J new 27L 132eL/. -~- CS7Yl
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where 8m is the mass added at free end 142 of cantilever 140. In order to
understand
the dependence of the minimum detectable mass to the cantilever design, the
expression should relate the minimum detectable mass to length 158, width 156,
thickness 148, density and modulus of cantilever 140 and the minimum
detectable
mass. So, subtracting equation (4) from equation (2) we have,
_~' __1 k _ k
f0 J new -
2?G Yl2e~. jy2ell + ~Yn
to simplify further, and rearranging to solved for 8m, we have
6
b'm=0.24~1~w~t~ p~ 1 z -1 ( )
Cl- 2~~f (0.98)l2 ~~
t E
which shows that for lower detectable mass, minimizing length 158, width 156,
density and the minimum detectable frequency would minimize the detectable
mass.
Thickness 148 of cantilever 140 largely affects the resonant frequency of the
system.
The resonant frequency, equation (2), can be expressed in terms of the
cantilever
properties as,
t E
'f° - 2TC(0.98)l2 P
which clearly indicates that, in order to keep the same resonant frequency
when
scaling down length 158 of cantilever 140 (which would scale down the minimum
detectable mass), thickness 148 needs to be scaled down as the square of the
scaling
factor. So for example to scale down the length 100 times, thiclaless 148
would need
to be scaled down 10,000 times in order to keep the same resonant frequency
for
cantilever 140.
The value of the resonant frequency will play a role for the detection of
the frequency. If the resonant frequency is too high, the deflection at fee
end 142 will
be very small, hence may not be detectable. Also circuitry 118 (Fig. 1) used
for
detection may not be feasible in the giga-hertz (GHz) range. However, keeping
the
resonant frequency too low is also not desirable, since this may cause
interference
with measurements from ambient noise. Thus the design of cantilever 140 should
be
made for the desired resonant frequency and minimum detectable mass, as shown
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graphically in Fig. 6 for exemplary cantilevers having width 156 of 1 ~,m and
various
thicknesses 148.
By way of example, for illustrative purposes, the exemplary
microfabrication and application of arrays of silicon cantilever beams as
nanomechanical resonant sensors according to the present invention, are
designed to
detect the mass of individual virus particles. The dimensions of the
fabricated
cantilever beams 140 are in the range of 4-5 ~,m in length 158, 1-2 ,um in
width 156
and 20-30 nm in thickness 148. The virus particles used as an analyte for the
exemplary embodiment were Vaccinia virus, which is a member of the Poxvi~idae
family and forms the basis of the smallpox vaccine. The frequency spectra of
cantilever beams 140, due to thermal and ambient noise, were measured using a
laser
Doppler vibrometer under ambient conditions. The change in resonant frequency
as a
function of the virus particle mass binding on the surface of cantilever beam
140
forms the basis of the detection scheme. A single Vaccinia virus particle has
an
average mass of 9.5 femto-grams (fg). The exemplary device can be very useful,
for
example, as a component of biosensors for the detection of air-borne virus
particles or
other analytes.
Known macroscale quartz crystal micro-balance devices for the
detection of virus particles require an external power supply and detection of
the
detachment of virus particles were measured (see, for example, M. A. Cooper,
F. N.
Dultsev, T. Minson, V. P. Ostanin, C. Abell, D. I~lenerman, Nature Biotechnol.
19,
833, 2001, incorporated herein by reference). The below discussed exemplary
embodiment according to the present disclosure includes nanomechanical devices
formed on an integrated circuit, with a measurement set-up sensitive enough to
measure thermal and ambient noise induced deflections and thus not requiring
an
external source to excite the cantilever beams (see, for example, B. Ilic, B.
Ilic, D.
Czaplewski, M. Zalalutdinov, H. G. Craighead, P. Neuzil, C. Campagnolo, C.
Batt, J.
Vac. Sci. Technol. B 19, 2825, 2001, incorporated herein by reference). In
order to
fabricate exemplary device 200, shown in various stages of fabrication in
Figs. 7A-E
and 8A-E, P-type (100) 4" silicon-on-insulator (SOI) wafers 202 are used as
the
starting material, shown in Figs 7A and 8A, for one exemplary fabrication
method.
The wafers have SOI layer 204 of 210 nm thiclcness and buried oxide (BOX)
layer
206 thickness of around 390 nm. Wet oxidation followed by buffered
hydrofluoric
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(BHF) etching is performed in order to thin SOI device layer 204 down to 30
mn.
Photolithography followed by reactive ion etching (RIE) using Freon 115 to
then etch
SOI layer 204 and CHF3/OZ in order to then thin BOX layer 206, in order to
pattern
cantilever beams 208 as shown in Figs. 7B and 8B.
After depositing a layer of plasma enhanced chemical vapor deposition
(PECVD) oxide as etch stop layer 210, etch window 212 was
photolithographically
patterned using BHF oxide etch, as shown in Figs. 7C and 8C. hl order to etch
the
underlying exposed silicon 204 and release cantilever beams 208, vapor phase
etching
using xenon difluoride (available from Xactix, Inc., of Pittsburgh, PA) can be
used,
resulting in device 200 as shown in Figs. 7D and 8D. After cantilever beams
208 are
released, oxide 206 is etched in BHF, rinsed in DI water, immersed in ethanol
and
dried using critical point drying (hereinafter sometimes CPD), resulting in
device 200
as shown in Figs. 7E and 8E.
Although measurement of the unloaded and loaded cantilever resonant
frequencies may be performed in many ways, one exemplary embodiment of system
220 associated with device 200 uses microscope scanning laser Doppler
vibrometer
222 (Model #MSV-300 available from Polytec PI of Auburn, MA) with a laser beam
spot size of around 1-2 pm. Vibrometer 222 may include, for example, monitor
224,
CCD camera 226, microscope 228, scanner controller 230, vibration controller
232,
oscilloscope 234, sensor head 235, laser 236, beam splitter 238, detector 240,
reference signal 242, and measurement signal 244. The resonant frequencies of
typical cantilever beams of length around 5 ,um, width around 1.5 ,um, and
thickness
around 30 nm are in the 1-2 MHz range with quality factor of around 5-7.
After device 200 fabrication, cantilevers beams may be cleaned in a
solution of H2O2:H2S04=1:1, rinsed in DI water, immersed in ethanol, and dried
using
CPD. The frequency spectra can be then measured in order to obtain the
'unloaded'
resonant frequencies of cantilever beams 208. Next, analyte, for example,
purified
Vaccinia virus particles in DI water can b a introduced over cantilever beams
208 and
allowed to incubate for 30 min, following which the cantilever beams are
rinsed in
ethanol and dried using CPD so as to minimize stiction of the cantilever to
the
underlying sufaces. The resonant frequency of cantilever beams 208 are then
measured again in order to obtain the 'loaded' resonant frequencies of
cantilever
beams 208 with the analyte.
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Using the mechanics of a spring-mass system, the added mass for the
corresponding change in resonant frequency can be determined. The change in
mass
(placed right at free end 142 of cantilever beam 140 shown in Fig. 4) in
relation to a
change in resonant frequency can be given as,
k 1 _ 1
dm = 4~t ~ ~ ~'a (7)
fl J o
where k is the spring constant of cantilever beam 208, fo is the initial
resonant
frequency, and fl is the resonant frequency after the mass addition.
Cantilever beams
208 can be calibrated by obtaining their spring constant, k, using the
unloaded
resonant frequency measurement fo, quality factor Q, and the plan dimensions
(length
and width) of the cantilever beam. The resonant frequency and the quality
factor can
be obtained by fitting the vibration spectra data to the amplitude response of
a simple
harmonic oscillator (SHO). The amplitude response of a simple harmonic
oscillator
(SHO) is given as,
fo (8)
A(f ) = Ad~
2 f2
f Z f a ~ + f ~ 2
where f is frequency in Hz, fo is the resonant frequency, Q is the quality
factor and
Aa~ is the cantilever amplitude at zero frequency, as described by D. A.
Waiters, J. P.
Cleveland, N. H. Thomson, P. I~. Hansma, M. A. Wendman, G. Gurley, V. Elings,
Rev. Sci. Instrum. 67, 3583, 1996, incorporated herein by reference). The
measured
spring constant of exemplary cantilever beams 208 is around 0.005-0.01 N/m.
Virus particles (shown as 132 in Fig. 2) can be counted by observing
cantilever beams 208 using a scanning electron microscope (hereinafter
sometimes
SEM). The change in frequency upon addition of mass can be detected by a laser
Doppler vibrometer 246, as shown in Fig. 9. The effective mass contribution of
the
viruses can then be calculated based on their relative position from the fixed
end of
the cantilever beams (for example, fixed end 144 of cantilever 140, shown in
Fig. 4).
Using the measurements from various cantilever beams 208, the resonant
frequency
shift (decrease) versus the effective number of virus particles observed on
the
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cantilever beam, can be plotted as shown in Fig. 10. The relationship can be
found to
be linear, as expected, to verify the validity of the measurements. Referring
again to
Fig. 3, the resonant frequency shift (~f = 60 kHz) after the addition of a
single virus
particle is shown. Fig. 3 shows a 60 kHz decrease in the resonant frequency of
a
cantilever beam having a plan dimension of L = 3.6 ~,m and W = 1.7 ,um. The
unloaded resonant frequency fo = 1.27 MHz, quality factor Q = 5, and spring
constant
k = 0.006 N/m. The resonant frequencies may be obtained by fitting the
amplitude
response of a simple harmonic oscillator to the measured data.
In one experiment using exemplary device 200 according to the above
fabrication and method, the average dry mass for a single Vaccinia virus
particle was
measured to be 9.5 fg, which is in the range of the expected mass of 5-8 fg
(see Bahr,
G.F., Foster, W.D., Peters, D. and Zeitler, E.H. Variability of dry mass as a
fundamental biological property demonstrated for the case of Vaccinia virions.
Biophys. J. 29:305-314, 1980, incorporated herein by reference). The measured
mass
sensitivity of exemplary cantilever beams 208 for a 1 kHz frequency shift is
160
attograms (ag) added mass (6.3 Hz/ag).
Another exemplary embodiment according to the present invention
integrates device 200 with on-chip antibody-based recognition and
concentrators, for
ultra-sensitive detection of air-borne virus particles, for example, device
100 shown in
Fig. 1.
Another exemplary micro-fabrication technique according to the
present invention may be used for fabricating device 300 having ultra-thin
cantilever
beams 302 in single crystal silicon with no stress. The exemplary process
utilizes a
technique called MELD (Merged Epitaxial Lateral Overgrowth) and can be
regarded
as an extension of selective epitaxial growth (SEG) and epitaxial lateral
overgrowth
(ELO) of crystalline material. SEG is a form of vapor phase epitaxy (VPE), and
is a
variation on the conventional full wafer epitaxy process known in the art. In
SEG, the
epitaxial deposition conditions are adjusted to prevent silicon deposition on
the
insulator region 304, for example, Si02, while silicon epitaxial growth occurs
only on
the exposed silicon in the seed windows 305 (see, for example, Bashir, R.,
Venkatesan, S., Neudeck, G. W. and Denton, J. P. A polysilicon contacted
subcollector BJT for a three-dimensional BiCMOS process. IEEE Electron Device
Letters 13:392-395, 1992, incorporated herein by reference). Referring to Fig.
11, if
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the selective growth is allowed to continue beyond the point where the
epitaxial
silicon is at level with the top of the insulator region, the silicon will
grow vertically
as well as laterally across insulator maslc 308 and under oxide layer 310,
forming
cantilever 302.
Cantilevers with thickness ranging from 0.2-0.5 Vim, maximum length
of around 130 ~m and widths of around 20 ~.m and 10 ~m have been fabricated
using
the above described method and known surface micro-machining techniques.
Mechanical characterization of device 300 can be performed by
measuring the resonance frequency using thermal noise to excite unloaded
cantilever
302 as shown in Figs. 13 and 14, and by adding known micro-sized particles 304
as
shown in Figs. 15 and 16. See also, known particles located at free end 306 of
cantilever 302 in Figs. 17A-D.Young's modulus, extracted from the added mass
approach was found to be in the range of 80-110 GPa and the mechanical quality
factor was measured to be in the range of 20-50 in air. Such cantilevers 302
can be
scaled to thickness of less than 100 nm and can be integrated into micro-
fluidic
channels (for example, channel 104 of Fig. 1 ) within the substrates for a
wide variety
of chemical and biological detection applications.
To determine the mechanical characterization of cantilever beams 302,
thermal mechanical noise is sufficient to oscillate cantilever beams 302 whose
deflections can be detected by an AFM that employ the optical lever technique
(see,
for example, Meyer, G. and Amer, N. M. Novel optical approach to atomic force
microscopy. App. Phys. Letters, 53:1045-1047, 1988, incorporated herein by
reference). The advantage of this method over driving the cantilever using a
piezoelectric or other exciter for mechanical characterization is that it does
not excite
other stiffer, higher mechanical resonance modes such as that of the
supporting
member to which cantilever 302 is coupled. In one exemplary system, the
cantilever
deflection signal was extracted from a Dimension 3100 SPM (see, for example,
Meyer, G. and Amer, N. M. Novel optical approach to atomic force microscopy.
App. Phys. Letters, 53:1045-1047, 1988, incorporated herein by reference),
using the
DI signal access module, and then digitized. The power spectral density
(hereinafter
sometimes PSD) of the signal was then evaluated using MATLAB software. The
thermal spectra data was then fit to the amplitude response of a simple
harmonic
oscillator (hereinafter sometimes SHO), using equation (8) above, where f is
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frequency in Hz. The quantities fo (resonant frequency), Q (the quality
factor), and
Aa~ (the cantilever amplitude at zero frequency) were extracted from the fit
of
equation 10 to the measured data.
The technique was also used to then determine the mechanical
properties such as stiffness (or spring) constant of the cantilever beams
using the
added mass (or Cleveland) method (see, for example, Cleveland, J. P., Manne,
S.,
Bocek, D. and Hansma, P. K. A nondestructive method for determining the spring
constant of cantilevers for scanning force microscopy , Review of Sci. Inst.,
64:403-
405, 1993, incorporated herein by reference) was used. For example,
polystyrene
spherical beads 308-314 of known mass, shown in Figs. 17A-D, were placed at
the
free ends of cantilever beams 302 using, a micromanipulator. Spherical beads
of
diameter of around 5.48 ~,m and 3.18 ~,m were used in the exemplary system.
Using
the density of polystyrene of p = 1.05 ~ 103 kg/m3, the masses of the beads
can be
calculated to be in the range of 90.5 picograms (pg) and 17.7 pg,
respectively. Due to
variation in the diameter of individual beads from the stated specifications
of the
manufacturer's values, the diameter can be measured using an optical
microscope.
The change in resonant frequency, fl, due to addition of a single mass, Ml, as
shown
in Fig. 19, can be measured and used to detect and extract the mass of the
bead 308.
The spring constant can be evaluated using,
(9)
k (2~z)2 (1/.fi )Z~l~l~.fo
If masses are added right at the free end 306 of cantilever beam 302,
giving
M = k(2~cf')-2 - m * , (10)
where, M is the total added mass.
It is also desirable to determine the minimum detectable mass that
could be measured by cantilever beams 302. Assuming the minimum detectable
frequency to be 6f = 1 Hz and setting fl = (fo - bf) in equation (9), it is
possible to
determine the theoretical minimum detectable mass. Table I, below, presents
the
values of the measured spring constant, effective mass and mass resolution for
previously microfabricated cantilever beams 320 and 330 shown in Figs. 21 and
22,
respectively.
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These masses were detected by measuring the vibrations of the
cantilever using optical means. Cantilever 320 shown in Fig. 21 was placed in
a
modified AFM system and the laser reflection off the cantilever surface was
detected
using a quad photo-detector, thus allowing us to measure the vibration of the
cantilever 320, for example, as shown for cantilever 302 in Figs. 13 and 15,
however,
other techniques to detect the vibrations may be used.
Table 1.
Planar dimensions and measured values of spring constant and
effective mass. Also listed are the extracted thickness from the effective
mass, the
extracted Young's modulus from the spring constant (assuming the given planar
dimension), and the ideal calculated value of mass resolution from equation
(9),
assuming 8f = 1 Hz for cantilever beams 320 and 3 30, shown in Figs. 21 and
22.
CantileverLength, MeasuredMeasuredExtractedExtractedCalculated
l
DesignationWidth, spring EffectiveThickness,Young's Mass
w
Constant,Mass, t Modulus,Resolution,
k m*
(N/m) (kg) ~l~m) E Om
(GPa) (g)
320 1= 78 0.145 4.99 0.49 100 1.17 ~
~m ~ 10-13 10-la
w = 23
~m
330 1= 129 0.0443 9.56 0.54 97.4 4.73 ~
~ 10-13 10-1a
~m
w=24
~m
As shown in Table 1, cantilevers 320 and 330 are capable of detecting masses
down
to about lOfg using a detection resolution of 1Hz change in frequency.
However, the
detection of such a small frequency change likely requires driving the
cantilevers
using forced excitations rather than thermal noise sources. For example, a
piezo-
electric film connected to an external signal generator to sweep the signal
frequency
and measure the resonant frequency of cantilevers 320 and 330 could be
utilized.
The design of cantilever beams is critical to the overall system design
and the ability to detect single virus particles or other minute analytes.
Using
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equations (6) and (7), the minimum detectable mass can be calculated as a
function of
the cantilever geometry, for example as shown in Fig. 6. As the length, and
correspondingly the mass, is decreased, the minimum detectable mass is
decreased.
To be able to detect single virus particles, for example, the size of a
cantilever would
need to be reduced to Sum wide x about l0um long. For these dimensions, the
resonant frequency would be larger than 1-5 MHz if a thickness of O.Sum is
used.
However, if the thickness is reduced down to 20mn, them the resonant frequency
is
also reduced to below 100kHz, which likely would be easier signal to detect
and
process.
There are at least three alternatives for the fabrication of the silicon
cantilevers, for example, silicon-based cantilevers which can included
integrated
piezo-resistive elements to provide an electrical output. One option, perhaps
the most
practical one, is to use commercially available S OI (silicon-on-insulator)
wafers,
wluch can have thicknesses down to O.lum (100nm). These wafers can then be
used
to fabricate surface micro-machined cantilevers. The main drawback of this
approach
is that the silicon material might have residual stress and the cantilevers
might be
curled and stressed upon final release. Another option is the use of the above
discussed technique using merged epitaxial lateral overgrowth (MELD) and
chemical
mechanical polishing (hereinafter sometimes CMP) of silicon. Yet another
fabrication option is 'tunnel epitaxy' shown in Figs. 11 and 12, which allows
the
fabrication of cantilevers down to a thickness of less than l Onm. In this
case, a tumlel
is defined using deposited films and selective growth of silicon is performed
to fill the
tunnel with single crystal material, forming an ultra-thin cantilever or other
suspended
structure. The films around the silicon can then be removed, thus releasing
these
nano-mechanical structures.
Another exemplary device 400, shoml in Fig. 23, according to the
present invention may, include piezo-resistive elements) or transistor region
elements) 404 integrated into suspended member 402. Element 404 may be used
for
the detection of the deflection, vibration, or surface stress or energy of
suspended
member 402. Element 404 can be grown selectively at the anchored or fixed end
406
where the stress is maximized, specifically, where suspended member 402 joins
support member 408 of device 400, especially at the surfaces of the junction
of
members 402 and 408. The growth of selective elements 404 enables a device
such
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as device 100 (Fig. 1) or the devices shown in Figs. 25A-D having an array of
cantilevers or other suspended members for detecting selective analytes.
In another exemplary embodiment of the present disclosure, using
standard integrated circuit and MEMS processing technologies, cantilevers or
other
suspended structures can be fabricated with lateral dimensions limited only by
the
resolution of the photolithography used, and thickness defined by thin silicon-
on-
insulator (SOn layers, formed by wafer bonding, separation by implanted oxygen
(SIMOX), or confined lateral selective epitaxial growth (CLSEG). These
cantilevers,
which typically have a mass of less than one nanogram (ng), have fundamental
resonant mode frequencies in the range of 100 kHz, and are easily stimulated
through
room-temperature thermal energy. By monitoring the resonant frequency of these
cantilevers, the attachment of particles with extremely small masses can be
detected,
and the total attached mass quantified.
By using on-chip signal processing and control, integrated piezo-
resistive or transistor detection methods using, for example, device 400 shown
in Fig.
23, eliminate the need for external components by electrically measuring the
surface
strain or other characteristic induced by deflection or vibration of
cantilever 402.
Referring to Fig. 24, in exemplary device 400 having a piezo-electric
element 404, the resistance of element 404 changes in response to strain,
which is
induced in cantilever 402 by deflection. When cantilever 402 is deflected, a
strain
gradient is induced throughout the body of the cantilever, with zero strain at
the
center. One surface is in tension (positive strain), while the opposite
surface is in
compression (negative strain). Since the response of piezo-electric element
404 to
strain is approximately symmetric about zero strain, an element that
penetrates
through the entire cantilever would not respond to deflection. The response of
the top
half of the element would be equal and opposite to that of the bottom half,
resulting in
no net response. Therefore, to be most effective piezo-electric element 404
must be
restricted to the near surface region of cantilever 402. While this is simple
to
accomplish by implantation and/or diffusion into relatively thick cantilevers,
it can be
very difficult to achieve in sub-100 nm thiclc cantilevers.
Alternative, exemplary device 500 (shown in Figs. 26A-B) having
transistor element 510 at the junction of cantilever 502 and support member
508
offers a possible solution to this limitation. For example, device 500 may
utilize
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ultra-thin channel 510 of field effect transistor (FET) 512, for example a
MOSFET
also having gate 504, source 514, drain 516, and source/drain region 51 l, as
a piezo-
electric sensing element. Conductive channel 510 of FET 512 may be confined to
within a few nanometers of the surface, thus in the region of maximum strain
upon
deflection of cantilever 502. Furthermore, it has recently been demonstrated
that the
mobility of the carriers in this channel 510 can be significantly enhanced by
tensile
strain, especially close to the surface of cantilever 502, as shown in Fig.
27. Thus the
combination of these two characteristics, piezo-electric resistance and
carrier
mobility, makes a device such as a MOSFET an advantageous deflection sensor
for
ultra-thin cantilevers.
Fig. 27 was generated for device 500 by simulation using SCRED 2.0,
a tool which self consistently solves the Schrodinger and Poisson equations.
The
structure simulated is a dual gate n-channel MOSFET with a background
concentration of 1016 cni 3, a body thickness of 10 nm, an oxide thickness of
1.5 nm,
and a metal gate with a work function of 4.6 eV. The back gate was held at a
constant
zero bias, while the top gate was swept from zero to two volts. As Fig. 27
indicates,
the channel carriers are contained within the top half of the structure, with
the peak
concentration occurring at approximately 1 mn from the oxide interface. This
level of
confinement should enable MOSFET-based piezoresistive detection in cantilevers
as
thin as 10 nm.
A MOSFET based strain sensor can be accurately located at this point
of maximum strain by using sidewall processing techniques. An exemplary
fabrication method for device 500 shown in Fig. 26A-B may be:
1. Begin with an SOI wafer.
2. Thin the SOI in the area where cantilever 502 will be formed
using an anisotropic etch. This forms an abrupt step between support member
50~ and
the top surface of cantilever 502.
3. Oxidize the silicon surface to form gate oxide 51 ~.
4. Conformally deposit polysilicon gate material 504.
5. Anisotropically etch polysilicon gate material 504 through the
entire deposited thickness. This leaves a sidewall of polysilicon on any
exposed
vertical surface, with a lateral width approximately equal to the deposited
thickness.
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6. Implant the source 514 and drain 516 regions appropriately,
using polysihicon gate 504 as an implantation mask.
7. Release cantilever 502 by wet or dry etclung.
Another potential advantage of using a MOSFET-based deflection
sensor such as device 500 is that by externally modulating the gate bias it
may be
possible to directly obtain the vibrational frequency spectrum of cantilever
504,
through a known technique, for example heterodyne mixing, or other known
signal
processing techniques. Heterodyne mixing uses the fact that the product of two
sinusoidah signals is the sum of two sinusoids with frequencies equal to the
sum and
difference of the original sinusoidal frequencies. By filtering out the sum
portion of
the signal, the result is a lower frequency signal with the same information
content as
the original signal. Heterodyne mixing is well know from radio receivers that
"mix
down" the incoming RF signal to an "intermediate frequency" that is more
easily
processed by downstream circuitry.
Another example of the use of mixing is the lock-in amplifier, in which
a reference signal is used to stimulate a device under test (DUT). The output
of the
DUT is then multiplied, or "mixed," with the reference signal, and
subsequently
filtered to remove the sum component. Assuming the response of the DUT is
linear,
the result will be a DC signal that is proportionah to the magnitude of the
transfer
function of the device at the reference frequency.
The small signal drain 516 current of MOSFET 512 operating in the
saturation region is
id (t) = gmvg (t) (11)
where g"1 is the transconductance of MOSFET 512, given at low frequencies by
gm =k~~(vc-yT) (12)
where k is a proportionality constant, ,u~ is the channel mobility, Tj~ is the
DC gate
voltage bias, and hT is the threshold voltage. Assuming that channel 510
mobility is
linearly dependent on the strain of cantilever 502, the transconductance will
also be
linearly dependent on this strain, through the channel mobility term.
Therefore the
transconductance can be written as
= k~ll~ 1 "~ ~S S(t) Y G - ~~. ) = 1 -f-' ~S iJ (t) gm0
gm (t) - k~c (t) ( G T ~ ~0 ~ ~0
(13)
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where g",o is the unperturbed transconductance, ~,S is the linear coefficient
of channel
mobility strain dependence, and S(t) is the strain in the channel 510 region.
Note that
the strain and therefore the transconductance are explicitly written as a
function of
time. ubstituting this expression for transconductance in drain 516 current
expression
yields:
id (t) = 1 + ~S S(t) g»~o~'g ct) = gmovg ct) + ~S grno"~ct)vg ct)
fro ~o (14)
The first term in equation (14) is the unperturbed response of
MOSFET 512 under the application of a small signal. The second term is the
mixing
term that is of interest in tlus discussion. The Fourier transform of (4) is
id c~) = g"~ovg c~) + ~'S g"~o [sc~) ~ vg (~)~
~~ (15)
If we assume that gate 504 voltage is pure sinusoidal with a frequency
of c~ o, then
vg (w)=vg ~/2~~(e~-~o)+~(ev+~o)) (16)
and
S(~)~vg(~) - f s(~)vgc~-~~dS2
- vg ~c/2(S(~-rvo)+S(c~+wo)) (17)
Substituting (7) into (5) yields
zdc~)=g~novgc~)+'~S g"~ovg ~~2(sc~'-~'o)+S'(ev+wo~)
~o (1 S)
Applying a low-pass filter with a cut-off frequency less than c~ o
eliminates the unperturbed response and the sum response (noting that
S (-ev) = S (~) ), leaving
id c« ~ 0) _ ~S g~novg ~'~2 S (wo ~ (19)
~o
Therefore the filtered response has a DC component that is
proportional to the amplitude of the vibrational strain at a frequency of w o.
By slowly
sweeping the frequency of the voltage applied to gate 504 the entire
vibrational
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frequency spectrum can be obtained directly. Note also that the result is
proportional
to the transconductance of MOSFET 512, wluch provides gain to amplify the
small
vibrations of cantilever 502 that are of interest.
In one alternative embodiment including FET 512, transistor element
S 510 is located on another suspended structure, such as a bridge, rather than
cantilever
502. In another alternative embodiment including FET 512, transistor element
510 or
511 located on cantilever 502 (or another form of suspended structure) may be
a gate
which cooperates with a channel located on another portion of device 500 such
that
the distance between the gate and channel as cantilever 502 deflects induces
changes
in the channel current.
Dielectrophoresis (DEP) may also be utilized in an exemplary
embodiment of the present invention. DEP is the translational motion of
neutral
particles in a non-uniform field region (see, for example, Pohl, H.A. The
motion and
precipitation of suspensoids in divergent electric fields, J. Appl. Phys. 22
(1951) 869-
871, incorporated herein by reference). DEP has been demonstrated to be able
to
capture and separate biological materials in fluid using micro-fabricated
electrodes
with ac electric fields (see, for example, Li, H. and Bashir, R.
Dielectrophoretic
separation of live and heat-inactivated Listeria on microchips. Sensors and
Actuators,
In press, 2002a, incorporated herein by reference). Neutral particles
(including, for
example, biological cells) become polarized due to the presence of electric
fields.
DEP forces can occur on cells when a non-uniform electrical field interacts
with the
field-induced electrical polarized particles, as shown in Fig. 28. The time-
averaged
DEP force F for a dielectric sphere immersed in a medium in constant field
phases in
space is given as:
~ - ~m 2
F = ~?C~O~ynY'3 Re ~ * DI E' yarns
~p -I- ~~m (20)
where Eo is the vacuum dielectric constant, r is the particle radius, E,.,,,5
is the root
mean square value of the electric field, and Ep* and Em* are the relative
complex
permittivities of the particle and medium, respectively (see, for example,
Pohl, H.A.
The motion and precipitation of suspensoids in divergent electric fields, J.
Appl. Phys.
22 (1951) 869-871, incorporated herein by reference). Depending on the
relative size
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of the dielectric constant of the particle with respect to the medium, the
particle can
exhibit positive or negative DEP.
Advantageously, according to the present invention, DEP may be
incorporated into exemplary device 600, as shown in Fig. 29. Suspended silicon
members, for example, opposed cantilever pair 602 and 604 are used for a dual
purpose: (1) as electrodes for DEP in order to capture air-borne or
aerosolized virus
particles and (2) as a mass sensor in order to detect the virus particles.
Cantilevers
602 and 604 are scaled down in size by the exemplary processes described above
to
provide the mass sensitivity necessary for detection of individual virus
particles.
When an ac electric field or another predetermined waveform is
applied to cantilevers 602 and 604, which may be electrically coupled to
electrodes
608 and 606, respectively, biological cells 616 and 618, or other analytes,
will be
captured at regions 614 with the largest gradient of the field.
Advantageously, the
largest gradient of the field is located at free ends 610 and 612 of
cantilever beams
602 and 604, respectively, as shown in Figs. 29 and 30, which, as discussed
above, is
also the preferred mass location for maximum change in resonance frequency for
cantilevers 602 and 604.
The technique of DEP and manipulation of analytes by electrical
forces provides a unique means to control the separation dynamics of
biological
agents and other particles. The method has numerous biological and medical
applications, e.g., identification and characterization of individual cells,
purification
of cell subpopulations from mixture suspension, etc., especially, for example,
with
integrated use of DEP separation and trapping of analytes such as air-borne
infectious
agents, combined with the detection of the analytes using micro-cantilevers.
Since the dielectric properties of different species of particles are
different and the dielectric constants of both particle and medium are
functions of
frequency, different species of particles at a given frequency may have
opposite DEP
responses, e.g., positive and negative DEP, respectively. By choosing a
predetermined waveform of proper frequency and a suspending medium so that two
different particles with different dielectric properties may experience
positive and
negative DEP respectively, a very useful method to selectively separate
particles of
different dielectric properties arises.
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Biological entities such as cells, proteins and DNA consist of adjacent
structures of materials that have very different electrical properties and
exhibit large
induced boundary polarizations that are highly dependent on the applied field
frequency as well as their physiological states. For example, the cell
membrane
consists of a very thin lipid bi-layer containing many proteins and is highly
insulating
with a conductivity of around 10-7 S/m, while the cell interior contains many
dissolved charged molecules, leading to a conductivity as high as 1 S/m. Upon
death,
the cell membrane becomes permeable and its conductivity can increase by a
factor of
104 due to the cell contents exchanging freely material freely with the
external
medimn through the small pores on the membrane. This large change in the
dielectric
properties on cell death indicates a large change in the dielectric
polarizability. Hence
a large difference in DEP responses (positive and negative respectively) and a
selective separation can be achieved between live and dead cells.
DEP is particularly useful in the manipulation and separation of
microorganisms and has been employed successfully in isolation and detection
of
sparse cancer cells, concentration of cells from dilute suspensions,
separation of cells
according to specific dielectric properties, and trapping and positioning of
individual
cells for characterization (see, for example, Wang, X., Huang, Y., Gascoyne,
P. R. C.
and Becker, F. F. IEEE Transactions On Industry Applications 33:660-669, 1997;
Huang Y, Holzel R, Pethig R, Wang X-B, Differences in the ac electrodynamics
of
viable and nonviable yeast-cells determined through combined dielectrophoresis
and
electrorotation studies. Phys. Med. Biol., 37:1499-1517. 1992; Markx GH, Huang
Y,
Zhou X-F, Pethig R (1994) Dielectrophoretic characterization and separation of
microorganisms. Microbiol. 140:585-591; Becker et al., 1994; and Stephens et
al.,
1996, incorporated herein by reference). Continuous separation can also be
achieved
by combining with a technique similar to field-flow-fractionation (see, for
example,
Markx GH, Huang Y, Zhou X-F, Pethig R (1994) Dielectrophoretic
characterization
and separation of microorganisms. Microbiol. 140:585-591; all of which are
incorporated herein by reference). Yet, the reports that deal with the
applications of
DEP in separation of viruses and small bacteria are rare. The first report of
molecular-scale particle manipulation was that of Washizu et al." 1990, where
they
reported DNA fragments of 48.5 kilobase pairs (or about 30 mega-daltons (mDa)
in
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molecular weight, which would be ~30 nm radius if the molecule were closely
packed
into a sphere were trapped and stretched (approximately 16~,m long) by
positive DEP.
A more recent work demonstrated that the same technique can be used
to precipitate DNA and proteins as small as 25 kilodalton (kDa) (see Washizu,
M.,
Suzuki, S., I~urosawa, O., Nishizaka, T., Shinohara, T., Molecular
dielectrophoresis
of biopolymers. IEEE Trans Ind Appl 30:835-843, 1994, incorporated herein by
reference). The step downward in size has been accelerated by advances in
fabrication technology such as the use of electron beam lithography, which
allows the
manufacture of electrodes with feature sizes of the order 100nm. Another study
demonstrated that molecules of the 68kDa protein avidin can be concentrated
from
solution by both positive and negative DEP (see Bakewell, D.J.G., Hughes,
M.P.,
Milner, J.J. andMorgan, H. Dielectrophoretic manipulation of Avidin and DNA,
Proc. 20th Ann. Int. Conf. Of the IEEE Engineering in Medicine and Biology
Society
(Piscataway, NJ: IEEE), 1998, incorporated herein by reference).
Attachment of the analytes, for example, proteins, on micro-fabricated
surfaces is important to the success of applications such as protein chips,
and the
attachment and capture of cells on micro-fabricated surfaces, such as the
cantilevers
of the Exemplary embodiments according to the invention. The attachment of
proteins on surfaces is complex when compared to the attachment of DNA to
surfaces. Proteins have to be attached in such a way that their structure and
functionality should be retained.
The attachment of antibodies and proteins has been demonstrated on
micro-fabricated surfaces using functional groups such as silane (see, for
example,
Britland, S., Arnaud, E. P., Clark, P., McGinn, B., Connolly, P., and Moores,
G.,
Micropatterning proteins and synthetic peptides on solid supports: A novel
application for microelectronic fabrication technology, Biotechnol. Prog. 8,
155,
1992; and Mooney, J. F., Hunt, A. J., McIntosh, J. R., Liberko, C. A., Walba,
D. M.,
Rogers, and C. T., Patterning of functional antibodies and other proteins by
photolithography of silane monolayers, Proc. Natl. Acad. Sci., 93(22), 12287,
1996,
all of which are incorporated herein by reference), amine (see, for example,
Nicolau,
D.V., Taguchi, T., Taniguchi, H., and Yoshikawa, S., Micron-sized protein
patterning
on diazonaphthoquinone/novolak thin polymeric films, Lahgmui~, 14(7), 1927,
1998,
incorporated herein by reference), carboxyl (see, for example, Williams, R. A.
and
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Blanch, H. W., Covalent immobilization of protein monolayers for biosensors
applications, Bi~senso~s and Bioelect~onics, 9, 159, 1994, incorporated herein
by
reference), and thiols (see, for example, Lahiri, J., Isaacs, L., Tien, J.,
and Whitesides,
G. M., "A strategy for the generation of surfaces presenting ligands for
studies of
binding based on an active ester as a common reactive intermediate: a surface
plasmon resonance study," 71, 777, 1999a, incorporated herein by reference).
Attachment of avidin on micro-fabricated surfaces using a bovine serum albumin
(BSA) layers has also been demonstrated in such a way that the avidin retains
its
binding ability to biotin and hence any biotinylated protein (see, for
example, Bashir,
R., Gomez, R., Sarikaya, A., Ladisch, M., Sturgis, J., and Robinson, J. P.
Adsorption
of Avidin on Micro-Fabricated Surfaces for Protein Biochip Applications,
Biotechnology and Bioengineering, 73, 4, 324-32~, May 2001, incorporated
herein by
reference). Patterning of ligands has also been demonstrated using
alkenethiolate
SAMs, which were produced on Au layers and then ligands such as biotin was
printed
on the SAMs using micro-contact printing (see, for example, Lahiri, J.,
Ostuni, E.,
and Whitesides, G. M., Patterning ligands on reactive SAMs by microcontact
printing, Laragmui~°, 15, 2055, 1999b, incorporated herein by
reference).
Proteins micro-arrays have been demonstrated where proteins were
immobilized by covalently attaching them on glass surfaces that were treated
with
aldehyde-containing silane reagents (see, for example, MacBeath, G. and
Schreiber,
S. L., Printing proteins as microarrays for high-throughput function
determination,
Science, 2~9, 1760, 2000, incorporated herein by reference). These aldehydes
react
with the primary amines on the proteins such that the proteins still stay
active and
interact with other proteins and small molecules. 1600 spots were produced on
a
square cm using robotic nano-liter dispensing where each site was about 150-
200um
in diameter. All of the above approaches take a protein and devise a technique
to
attach it to a micro-fabricated surface by functionalizing one end of the
protein with
chemical groups that have affinity to that particular surface.
Regarding binding on antibody to silicon suspended members, the
silicon cantilever surface will always form a native oxide layer, on which the
antibodies will need to be attached. We have demonstrated the adsorption of
the
protein Avidin on patterns of silicon dioxide and used of fluorescent
microscopy to
detect binding of biotin (see Bashir, R., Gomez, R., Sarikaya, A., Ladisch,
M.,
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Sturgis, J., and Robinson, J. P. Adsorption of Avidin on Micro-Fabricated
Surfaces
for Protein Biochip Applications, Biotechnology and Bioengineering, 73, 4, 324-
328,
May 2001, incorporated herein by reference). The silicon dioxide microchip was
formed using plasma enhanced chemical vapor deposition while platinum was
deposited using radio-frequency sputtering. After cleasung using a plasma arc,
the
chips were placed into solutions containing Avidin or bovine serum albumin.
The
Avidin was adsorbed onto the microchips from phosphate buffered saline or from
PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto
bovine serum albumin (BSA) coated surfaces of oxide and platinum. Fluorescence
microscopy was used to confirm adsorption of labeled protein, or the binding
of
fluorescently labeled biotin onto previously adsorbed, unlabeled Avidin. When
labeled biotin in PBS is presented to Avidin adsorbed onto a BSA coated
microchip,
the fluorescent signal was significantly higher than for Avidin adsorbed onto
the
biochip alone. The results show that a simple and low cost adsorption process
will
deposit active protein onto a chip in an approach that has potential
applications in the
development of protein biochips for the detection of biological species.
In one exemplary embodiment, biotinylated BSA, which adsorbs
strongly onto a C18 modified silica surface at pH 7.2 through hydrophobic
interactions, has been used for analyte specific bonding. Its activity is
maintained, as
indicated by the strong adsorption of streptavidin, which was validated
through
fluorescence microscopy (see, for example, Huang, T., Gaba, A., Gomez, R.,
Bashir,
R., Sturgis, J., Robinson, J. P., Ladisch, M. R., Submitted to Biotechnology
and
Bioengineering, 2002, incorporated herein by reference). Subsequent capture of
fluorescently labeled biotin by streptavidin indicated that biotinylated
antibody could
be successfully attached to the surface. Recent binding studies indicate the
biotinylated BSA has low non-specific binding to IgG antibody and Liste~ia
cells
while streptavidin alone binds both L. ynohocytogeyaes and L. inraocua.
However,
when the streptavidin is blocked with BSA, the non-specific adsorption is
significantly reduced. Then when biotinylated antibody is fixed to the surface
specific binding of L. mofaocytogeyaes occurs.
Attaclnnent of proteins to silica (Si02) surface of chips has carried out
(see, for example, Mooney, J. F., Hunt, A. J., McIntosh, J. R., Liberko, C.
A., Walba,
D. M., Rogers, and C. T., Patterning of functional antibodies and other
proteins by
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photolithography of silane monolayers, Py~oc. Natl. Acad. Sci., 93(22), 12287,
1996,
incorporated herein by reference) where the functionality of biotinylated goat
antibodies was demonstrated using fluorescently labeled mouse immunoglobulin
IgG
as reporter molecules. The Si02 surfaces are first derivatized with
octadecyltrichlorosilane (ODTS) to form a C1$ surface. Biotinylated bovine
serum
albumin (BSA) is then adsorbed onto a Cl8 modified hydrophobic silica surface.
After washing the streptavidin can be adsorbed onto the biotinylated BSA. The
role
of streptavidin is to capture biotinylated monoclonal antibody and orient it
in a way
that enables the antibody to contact and capture Liste~ia mofzocytogehes.
While there are many covalent attachment schemes for biotin or
streptavidin, an exemplary embodiment according to the present disclosure
addressed
a sandwich scheme for the Cl8 modified surface using non-specific binding of
Listeria
monocytogeraes and Liste~ia monocytogenes binding to biotinylated antibody c1
1e9
using PBS buffer at pH 7.2 and incubated for 2 hours, the comparisons for
which are
shown in Fig. 31.
An additional exemplary embodiment according to the present
disclosure includes covalent attachment techniques of Immobilizing IgG C11E9
antibodies on a silicon dioxide surface for use in a biosensor to detect
capture of
pathogen Listeria monocytogenes (see, for example, Gaba, A., Sturgis, J.,
Robinson,
J. P., Gomez, R., Bashir, R., Ladisch, M. R. Immobilization of IgG C11E9 on a
silica
surface for use in a biosensor to detect capture of pathogen Listeria
monocytogenes,
in Press 2002, incorporated herein by reference). Thus, the development of a
platform for placing more selective antibodies for L. mohoc~togenes and other
target
cells has been demonstrated and can be extended to the detection of virus
particles.
While the physiology of living cells and viral particles are obviously
different, the
basic fabrication steps are similar. In both cases non-specific adsorption
must be
blocked, so that binding to the antigen is not masked by binding of similar,
non-target
species to the cantilever's surface itself.
Various surface derivatization approaches can be utilized to anchor
coronavirus specific antibodies to silicon cantilevers. For example, utilizing
several
heterobifunctional cross-linlcers and micro-patterning of cantilever surfaces
with the
coronavirus specific antibodies. Although exemplary cantilevers according to
the
present disclosure include well depths of about 14-16 ~,m, the problem of
suction
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could occur and hence surface modifications can be introduced to minimize such
stiction of cantilevers to substrate after the BHF etching of the oxide
encompassing
the cantilevers. The exemplary embodiment utilizes a coating including
hydrophobic
self assembling monolayers (SAM) films. For example, 1.0 mM of
octadecyltrichlorosilane (OTS) in 2,2,4-trimethylpentane (isooctane) may be
used as
the solvent to form the SAM coatings. Other anti-stiction methods and surface
modifications may alternatively be utilized.
Another exemplary embodiment utilizes covalent attaclunent
techniques of Immobilizing IgG Cl 1E9 antibodies on a silicon dioxide surface
for use
in a biosensor to detect capture of pathogen Listeria monocytogenes (see Gaba,
A.,
Sturgis, J., Robinson, J. P., Gomez, R., Bashir, R., Ladisch, M. R.
Immobilization of
IgG C11E9 on a silica surface for use in a biosensor to detect capture of
pathogen
Listeria monocytogenes, in Press 2002, incorporated herein by reference).
Thus, a
platform for placing more selective antibodies for L. monocyt~genes and other
target
cells may be extended to the detection of virus particle. Eliminate the non-
specific
adsorption of the virus particles in crucial. Therefore, anti-fouling or
blocking layers
such as BSA and other bio-chemical layer may be utilized. Additionally, DEP
forces
for the movement and manipulation of the virus particle may be utilized, if
they are
non-specifically adsorbed on the antibody coated cantilevers. An ac signal or
other
waveform may be pulsed at these electrodes in such a way that could
effectively
sweep the virus particles away from or toward the cantilevers, as desired.
At resonance, the vibrations of the cantilever could also provide a
novel method to detach the biological entities captured on the cantilever beam
surfaces. The non-specifically bound species could be detached first, for
example, at
approximately 0.1-1.0 pN when an anti-fouling agent is utilized, while the
specifically
bound entities will not be removed, as shown in Fig. 32. As the magnitude of
the ac
signal or other wave form is increased, the amplitude of vibrations will also
increase,
resulting in the release of specifically bound entities, for example, at
approximately
200 pN.
An overall system device, for example device 100 shown in Fig. 1,
may utilize many of the above described exemplary embodiments, including those
further described below. Additionally, as shown in Fig. 33, antigen-binding
sites,
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antibodies, linking molecules, and blocking agents may be used in conjunction
with
the suspended member in order to functionalize it for a specific entity.
Referring again to Fig. 1, device 100 may be micro-fabricated with
input port tube 124 with mechanical filter 120 that only lets particles less
than 0.5 um,
for example, pass through. Once particles are in the air stream in fluid
channel 104 of
integrated circuit 102, and they will contact DEP potential wells from the
particle
sorters 112 and 114, where they would get diverted into the appropriate fluid
channel
chambers having an array of cantilevers 106-110. Analytes with different
dielectric
properties or size will pass through the first sorter 112 and can be selected
by the
second sorter 114. Only one sorter 112 and 114 and one cantilever chamber may
also
be used. Cantilevers 106-110 may be coated with antibodies for specific virus
or
other analytes and will therefore capture these specific analytes. Another DEP
filter
116 after the cantilevers can be used to confine or concentrate the analytes
in the
region close to cantilevers 106-110 to maximize capture by cantilevers 106-
110.
Optical or electrical measurement of the resonant frequency change can detect
the
binding of single virus particles, for example, electrical measurement
utilizing signal
processor and control circuitry 118 implementing any of the above discussed
embodiments for electromechanical sensing. Mesh filters 122 at the output port
of
device 102 is useful to ensure that there is a mechanism to contain analytes
inside
device 102 if infectious agents are found in the air sample.
HEPA type filters and the rotary rod method for capturing viral
particles that are in the range of O.Sum or less in size my be used to
concentrate
analytes. For exaanple, an exemplary apparatus for validating the inventive
concept
may consist of a closed plastic box where known quantities of a virus can be
introduced into the air, and the air then circulated over the filter or
capture device.
The selection of the type of blower which will push the air across the filter
will be
important as will the size of the plexiglass box, which may be initially
fabricate to
contain half a cubic foot of air.
Viruses are generally 0.05-0.1 microns and hence, the exemplary
apparatus may use a surrogate viruses (that are not pathogenic) and focus on
particle
sizes in the 0.05-0.1 micron range. Calibration of the system may be done
using inert
particles (that are typically used for calibrating particle size instruments)
in order to
begin to identify optimal linear velocity through the filter/membrane, and
determine
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the effectiveness of particle capture. Operation using surrogate virus may use
standard culture methods. In addition, a labeling compound and fluorescence
microscopy can be used to identify virus that are trapped in the filter or
membrane.
Air-spray with aerosolized virus particles may also be directly injected into
device
100 at controlled rates for the purpose of performing proof of concept
validation.
Airborne microorganisms (bacterial, fungal spores, viral particles and
pollen) are known to cause various health effects, including infection,
hypersensitivity, toxic reactions, irritations, and inflammatory response
(see, for
example, Agranovski, I. E., V. Agranovski, T. Reponen, K. Willeke, and S. A.
Grinshpun, Development and Evaluation of a New Personal Sampler for Culturable
Airborne Microorganisms, Atmospheric Environment, 1-10, 2001, incorporated
herein by reference). Virus particles can range from 0.05-0.1 ~m in size and
are
highly resistant to extreme environmental conditions. To detect a few harmful
spores
from the air in a regular-sized office room represents a very challenging
technological
endeavor. The airborne particles need to be first pre-processed in order to
isolate the
bio-aerosol. The air samples in a room need to be pre-processed in order to
isolate the
airborne microorganism. A coarse filter can be used to filter out the larger
airborne
particles (see, for example, Battarbee, J. L., N. L. Rose, and X. Long, "A
Continuous,
High Resolution Record of Urban Airborne Particulates Suitable for
Retrospective
Microscopical Analysis," Atmosplae~ic Envi~orarnent, 31 (2), 171-181, 1997,
incorporated herein by reference). However, many other non-biological
particles also
have ~m-size characteristics. There are several methods including cylindrical
traps,
impingers, and centrifugal type collectors for filtering or sorting particles.
Cylindrical
traps worlc by coating the inner surface of a cylinder fist with a sticky film
such as
cellulose. Then, the pre-processed air is feed onto the surface. The viral
capture
efficiently is heavily influenced by the airflow velocity (see, for example,
Griffiths,
W. D., I. W. Stewart, S. J. Futter, S. L. Upton, and D. Mark, "The Development
of
Sampling Methods for the Assessment of Indoor Bioaerosols," J. Aerosol Sci.,
28(3),
437-457 (1997); Maus, R., and H. Umhauer, Collection Efficiencies of Coarse
and
Fine Dust Filter Media for Airborne Biological Particles, J. Aef°osol
Sci., 28(3), 401-
41 S (1997); and Mullins, J., and J. Emberlin, Sampling Pollens, J. Aerosol
Sci., 28(3),
365-370, 1997; all of which are incorporated herein by reference).
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In the impactor technique, a microscope slide is used as the standard
sampling surface in the volumetric viral trap developed by Hirst. In Hirst's
sampler,
the pre-processed air is drawn through a slit with the same dimensions as the
second
stage of the cascade impactor. The particles then contact the sticky surface
of a glass
slide, moving past the orifice at 2 mm h-1. Thus, over the course of 24 hr, a
trace that
is 48 mm long is obtained with the deposit at any point representing the mean
concentration over a 1 hr sampling period. This sampler has been adopted as
the
standard for most pollen sampling networks in Europe (see, for example,
Mullins, J.,
and J. Emberlin, Sampling Pollens, J. Aerosol S'ci., 2~(3), 365-370, 1997,
incorporated herein by reference). The rotary sample method was devised by
Perkins
(1957) for short-term sampling and is available through Aerobiology Research
Laboratories of Nepean, Ontario, Canada. In this sampler, airborne spores are
impacted onto the leading edges of a 'U'-shaped rod made of 1.6 mm square
cross-
section brass rod, with arms 6 cm long and 8 cm that are rotated through air
at 2500-
3000 rpm.
Advantageously, DEP filters or valves, which are selective to any
object having a dielectric constant different from the medium, for the capture
of
microorganisms may be utilized instead of the above mentioned approaches. For
example, polystyrene beads can be separated from buffer, and cells or spores
can be
separated from water (see, for example, Li, H. and Bashir, R.
Dielectrophoretic
separation of live and heat-inactivated Listeria on microchips. Sensors and
Actuators,
In press, 2002a; Li, H. and Bashir, R. Dielectrophoretic separation of live
and heat-
inactivated cells of Listeria on microfabricated devices with interdigitated
electrodes,
Proceedings of the Spring MRS 2002b. San Fransisco, CA; and Gomez, R., Bashir,
R., Bhunia, A.I~. and Ladisch, M.K. "Microfabricated device for impedance-
based
detection of bacterial metabolism", Proceedings of the Spring MRS 2002. San
Francisco, CA; all of which are incorporated herein by reference). This filter
trap can
be switched on/off by the ac fields and does not clog, unlike mechanical mesh-
like
filters. These filters can be placed at the end of the fluidic detection
chambers as
showxn in Figs. 34 and 35.
DEP separation of live and heat-treated Liste~ia ifayaocua cells has been
achieved as shown in Figs. 36A-C. This was the first report of separation of
Liste~ia
in water by DEP with about 90% efficiency by application of a 1V and SOI~Hz
signal.
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It was observed that the DEP behaviors of live 162 and dead 164 cells differed
in the
frequency range from ~30KHz to ~100I~HHz in the selected medium (water). Both
live
162 and dead 164 cells can collect either on the top centers of the electrodes
160 in
negative DEP, shown in Fig. 36A, or at electrode 160 edges in positive DEP,
shown
in Fig. 36B, for the special electric field configuration of the
interdigitated
microelectrodes used. As shown in Fig. 36C, at 50 kHz, live cells 162 located
to
electrode 160 edges and dead cells 164 located to electrode 160 centers. The
viability
of the cells was verified by a rapid epifluorescence staining method using the
LIVE/DEAD Bacterial Viability I~it (BacLight, available from Molecular Probes
of
Eugene, OR) and the live 162 and dead 164 cells can be monitored
simultaneously in
the experiments. It is envisioned that the separation and manipulation of
microorganisms aald viruses on biochips using DEP might become very useful
method
in sample preparation and preprocessing and in diagnostic applications.
Refernng to Figs. 37A and 37B, an exemplary device may utilize DEP
filter 700 to selectively capture a particle of interest inside the integrated
circuit.
Polystyrene beads 704 (coated with antibodies selective to target species) are
flowed
through the integrated circuit. An array of electrodes 702 may be used to
generate an
AC electric field at a frequency on the order of 1 MHz, and a peak intensity
of at least
106 V/,um. When beads 704 approach electrodes 702, they will experience a DEP
force which repels them away from regions where the gradient of the electric
field is
maximum (at the edges of electrodes 702). If this force is equal to or larger
than the
drag force exerted on beads 704 by the liquid flow, beads 704 remain trapped
in the
chamber (along with the bacterial cells they carry) while everything else in
the sample
flows out of the chip. Fig. 37A shows 2.38 ,um (diam.) beads 704 flowing
freely
through the chip while the electric field is off. Fig. 37B shows beads 704
accumulating in the middle of electrodes 702 when the field is turned on
(beads 704
cannot cross the edges of electrodes 702, where the DEP force if maximum).
The techniques described above can be used to concentrate bacteria,
cells, viruses, DNA, or proteins, as long as their dielectric constant is
different than
the dielectric constant of the medium that they are suspended in. Hence, this
technique can be used to separate virus particles from other particles in air,
or used to
concentrate particles of a particular type in a micro-chamber of interest.
Fig. 38
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shows an optical image of a device (0.75cm x 0.75cm), which was used to
perform
the above-mentioned studies of cells and beads in water and buffers.
Exemplary interdigitated microelectrodes according to the present
invention can be manufactured on silicon substrate using standard
photolithography.
The electrode material can be gold, 1000 A thick, magnetron sputtered onto a
100 A
thick seed layer of chromium. The width of the electrodes and the spacing
between
two adjacent electrodes can be simulated so as to produce enough DEP force to
stop
the particle in the air-flow. For proof of concept validation, the electrodes
can be
connected to an arbitrary wavefonn generator as the AC signal source by
attaching
two conducting wires to the contact pads. Fluorescently labeled aerosolized
samples
can be allowed to enter into the DEP chambers and a sinusoidal signal with
varying
voltage and frequency values can be applied to the electrode array. The
electrokinetic
behaviors can then be viewed on a TV monitor through a CCD digital camera on a
fluorescence microscope. The filters can be designed using ANSYS or other
finite
element software, which will allow the modeling of the ac fields as a function
of the
electrode geometry and spacing. The DEP forces will be calculated.
Another exemplary device 700 according to the present invention
includes an ultra-thin suspended silicon member 702, for example, a nano-wire,
providing an active resonant sensing area, for example a transistor channel
(or gate) in
the case of device including a field effect transistor. The suspended member
702 may
be grown, for example, by tunnel epitaxial growth, where the growth path of
the
silicon is restricted and is allowed only in one direction in a cavity. During
the
formation of the cavity, an oxide layer forming a roof of the cavity can be
made to
collapse due to stiction and subsequent growth of silicon results in very thin
single
crystal silicon wires suspended between two islands, which may form the source
and
drain of the FET.
Alternatively, by not doping the source and drain regions, the nano-
wires 'forming suspended members 702 can be simply used as resistors.
Resistive
nano-wires provides detection of molecules binding on the wire by measuring
resistance across the nano-wire.
By oscillating the nano-wire member 702 using the FET gate, the
current through the FET, specifically the FET channel formed by members) 702
is
modulated as a function of the distance between the suspended member 702 and
the
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gate. Also, the current will be function of the gate bias, as for typical
FETs. The
piezo-resistive effects on oscillating nano-wire members 702 also affect the
modulation of current due to stress changes that the nano-wire member
experiences
from mechanical oscillating. By monitoring the drain-source current through
the
FET, the amplitude of the oscillations and the frequency can be determined,
with high
signal levels and sinal-to-noise ratios compared to pure capacitative sensing.
Because the oscillating frequency and amplitude is sensed electically,
and high signal-to-noise ratios are expected, very small changes in resonant
frequncy,
due to the added mass of particles on nano-wire members 702 can be resolved by
incorporating feedback.
For a single crystal silicon (for example, E = 162 GPa and
density=2.33g/cm3) nano-wire member, having a length of S um, width of 3 um,
and
thickness of 0.01 um, the resonant frequency is 2.123340 MHz. Assuming a
minimum detectable frequency of 1 Hz, the minimum detectable mass is estimated
to
be, S.SxlO-l~g, which corresponds to the approximate mass of a single protein,
therefore smaller than the mass of a single virus or cell.
Alternatively, the novel device can be operated in other modes of
electrical operation and/or mass detection, for example, as resistors or
transistors,
with detection of a change in surface potential or a change in mass.
If a resistor is fabricated, then binding of entities on the surface of the
nano-wire members 702 will result in change in electrical resistance of the
resistor
due to change in surface properties. Members 702 can be thermally excited and
the
mechanical resonance can be measured electrically using capacitive technique
and the
bottom gate. Then once a mass is added (by a binding event, for example as
discussed for exemplary embodiments above), the resonance frequency will
change.
The resonance could also be measured using the changes in resistance value of
the
nano-wire member. Alternatively, nano-wire 702 can be capacitively excited
using
the bottom gate and the mechanical resonance can be measured electrically
using the
changes in resistance value of the nano-wire upon mass addition to the nano-
wire
member.
If device 700 includes an FET, then binding of analytes on the surface
of the suspended nano-wire members 702 will result in change the source/drain
current due to changes in surface potential.. Once the suspended nano-wire
member
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vibrates (thermal or capacitive excitation), the piezo junction effect will
change the
source drain current and that could be electrically detected. In addition, the
thermal
vibrations can also be detected capacitivehy using the bottom FET gate.
Alternatively,
the suspended nano-wire member 702 can be driven into resonance using the
bottom
S gate and the changes in source/drain current can be measured as a means to
detect the
vibration and thus change in resonant frequency, as discussed above for
cantilever
devices.
Although carbon nano-tubes and silicon nano-wires have been
demonstrated as single molecule biosensors, the fabrication methods that have
been
used for creating those devices are typically not compatible with modem
semiconductor manufacturing techniques and their large scale integration is
problematic. The exemplary method of fabrication of silicon nano-wires at
precise
locations described below overcome those limitations. The exemplary method of
fabrication allows for the realization of truly integrated sensors capable of
production
of dense arrays. Sensitivity of these devices to changes in the ambient gas
composition has also been demonstrated.
Minaturization of biological and chemical analysis tools to the level of
the "lab on a chip" decreases the analysis time and the sample size needed for
specific
detection in genomic and proteomic applications as well as in detection of
warfare
agents and environmental pollutants. In general, as the sensor dimensions
shrink
down to the size of the analyte, the sensitivity of the device increases.
Specifically,
suspended nano-wire type sensors are very attractive because their large
surface area
to volume ratio results in high sensitivity. Carbon nano-tubes and silicon
nano-wires
have been demonstrated as single molecule biosensors (see, for example, I~.
Besteman, J.L. Lee, F.G.M. Wiertz, H.A. Heering, C. Dekker, Naho Lett., 3,
727(2003); Y. Cui, Q. Wei, H. Park, C.M. Lieber, Science, 293, 1289, 2001,
incorporated herein by reference), but the fabrication methods that have been
used for
creating these devices are typically not compatible with modern semiconductor
manufacturing techniques and their large scale integration has been quite
problematic
(see, for example, J.F. I~lemic, E. Stern, M. Reed, Nature BioteclZnol., 19,
924, 2001,
incorporated herein by reference). However, the exemplary fabrication method
according to the present invention and initial fabrication test results on a
silicon nano-
wire sensors using top-down microelectronics processing techniques overcomes
the
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former problems. A process known as confined lateral selective epitaxial
growth
(CLSEG) (see, for example, P.J. Schubert, G.W. Neudeck, IEEE Elec. Dev. Lett.,
1l,
181, 1990, incorporated herein by reference) was utilized to obtain single
crystal
silicon nano-plates that are as thin as 7 nm and nano-wires as small as 40 nm
in
diameter at precise locations. The exemplary fabrication method allows for the
realization of truly integrated dense array of sensors. Initial testing of the
devices
showed sensitivity towards oxygen ambient, suggesting the possibility of using
these
sensors for chemical and biological detection.
Fabrication of exemplary device 800 may be performed on p-type low
doped silicon wafers 802. A 2000 ~ thick oxide 804 can be grown by wet
oxidation
for substrate isolation. A sacrificial layer 806 of amorphous silicon with 100
~ of
thickness can next be deposited and defined lithographically on the silicon
dioxide
layer 804, as shown in Fig. 40A. Another 40001 thick oxide layer 810 can then
be
deposited using plasma enhanced chemical vapor deposition (PECVD). Via holes
808 that will subsequently be filled with silicon and act as the FET source
and drain
regions, can be etched using a reactive ion etch, as shown in Figs. 40A and
41A.
The sacrificial layer of polycrystalline silicon can then be removed
selectively by wet etching using tetra-methyl-ammonium-hydroxide. The removal
of
the sacrificial layer defines a gap between the thermally grown oxide and
deposited
oxide. Due to the surface tension of the liquid after the rinse step, bridge
formed by
the top oxide collapsed, leaving via holes 812 near the anchors of the oxide
bridge
814, as shown in Fig. 41 C. Via holes 812 are then used as a mold for the
epitaxial
silicon to grow through which will later form the suspended nano-wire members.
A
seed hole 816 was wet etched in thermal oxide 804 on the source side down to
the
silicon surface 802, as shown in Fig. 40D, in order to grow epitaxial silicon
by a
CLSEG with no intentional doping.
The exemplary fabrication process yields good quality single crystal
silicon with low n-type doping (~ 1 O16 Cm 3). Epitaxial silicon grows through
via
holes 812 at the edges of the collapsed oxide bridge 814 as well as at the
interface of
the collapsed region. The silicon grown in the interface region between via
holes 812
forms a 6-7 nm thick plate. The excess silicon remaining on the surface can be
removed by chemical-mechanical polishing as shown in Figs. 40E and 41E. A high
dose n-type blanket implant can be performed in order to form conductive
source and
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drain regions 820 and 822, respectively. After depositing a 2000 A thick PEVCD
oxide insulation layer a high temperature anneal can be performed to activate
and
drive in the implanted dopant, and to densify the PECVD oxide. Contact holes
can
next be wet etched in the oxide to access the silicon source and drain regions
820 and
822. A 200 A of chromium, followed by 2500 A of gold was evaporated and
patterned to define electrical contacts, as shown in Figs. 40E and 41E.
The final step in the exemplary fabrication process is to uncover (thus
suspending) the silicon nano-wire members 824 by removing the encapsulating
oxide
by un-patterned wet etching in buffered hydrofluoric acid, shown in Fig. 40F.
The
fabrication method yields a film of silicon about 7 nm thick in the collapsed
regions,
and 50 nm diameter suspended nano-wire members s at the edges of the film
between
the anchors. Etclung in BHF for 6 minutes removes the oxide covering on the
device,
leaving the plate and wires in place, with a supporting oxide layer below, as
shown in
Fig. 45. A longer BHF etch (14 min) removes all remainng oxide as well as the
thin
silicon film 823 (Fig. 45) between the wires, resulting in the formation of
suspended
nano-wire members 824 (shown in Fig. 46, and shown in Fig. 39B as 702). After
rinsing, the samples can be soaked in methanol without drying to ensure the
complete
displacement of the water, and then air dried.
Experiments have been performed on both the plate and wire structures
in order to verify the feasibility of using the structures as field effect
sensors. While
fluidic detection of chemical and biological analytes is conceived, gas phase
measurements were initially performed due to simpler experimental setup. All
measurements were performed in a closed chamber where the ambient gas
composition and pressure was controlled. A 1 l~Hz 10 mV peak-to-peak
sinusoidal
probe signal superimposed on a variable DC bias was produced by a function
generator (Model DS345, available from Stanford Research Systems, of
Sumiyvale,
CA) and fed into the source electrode of device 700. The resulting drain
current was
amplified through a low-noise current preamplifier (Model SR570, also
available
from Stanford Research Systems) and detected using a lock-in amplifier (Model
SR850, also available from Stanford Research Systems). Dry nitrogen was used
to
purge the test chamber, and 20% oxygen in argon was used as the analyte gas.
Using
the described setup, we were able to directly collect the small signal
conductance
(dI/dV) of the device as a function of time and DC bias. The effect of ambient
gas on
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the conductance of the devices was investigated. As control measurements, we
tested
devices that were not released and found no reaction to oxygen ambient.
Released
devices exhibited up to a 9% decrease in conductance when exposed to 20%
oxygen
in argon.
The decrease in conductance of the wire is believed to be attributed to
the physiosorption of oxygen species on the nano-wire members resulting in a
decrease of the work-function of the silicon surface. It is known that the
work-
function of the silicon surface reduces upon exposure to oxygen, while it does
not
change upon exposure to inert gases. A decrease in the work-function will
result in
an increased energy barrier between the heavy doped n-type contact regions and
the
low doped wire, and result in a reduction of the current through device 700.
The
oxygen molecule, which is a diradical and very reactive, effectively induces a
net
negative charge on the surface of the wires. This results in a net depletion
of the
silicon nano-wire/native-oxide interface, causing an effective decrease of its
electrical
diameter. These hypotheses are confirmed by measurement results. Control
experiments were also performed on released devices with pure argon gas to
ensure
the conduction change was indeed due to oxygen. Devices which responded to the
argon-oxygen mixture did not respond to pure argon.
It was also seen that the decrease in conductance was reversible and
repeatable. Fig. 42 shows a decrease in the conductance of the plates upon
exposure
to oxygen, and recovery of the conductance after purging in nitrogen. However
it can
also be seen that the baseline conductance of device 700 shifts for the same
amount of
recovery time, indicating some irreversibility of the adsorption. In order to
fully
recover the conductance of devices 700, they were heated in vacuum at elevated
temperatures (80-90°C). A similar set of experiments was also performed
on the
nano-wire members 702. The nano-wire members 702 showed similar response to
exposed oxygen, however simply purging the test chamber was not sufficient to
recover the conductance of the wire device 700. Device 700 conductance
continued
to decrease at a slower rate after the nitrogen purge was started until the
chamber was
evacuated. After evacuation of the chamber the conductance stabilized at a
constant
value. In order to desorb the oxygen, the nano-wire device 700 was heated up
to 80°C
in vacuum and cooled back to room temperature. Upon this procedure device 700
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conductance increased due to desorption of oxygen gas from the wire surface. A
device cycling experiment is shown in Fig. 43.
The results obtained from gas phase measurements are very
encouraging, and suggest that the use of a top-down nano-fabrication technique
as
disclosed above is capable of producing nanoscale sensors for the detection of
very
low concentrations of analytes. By analyte specific receptors or ligand
functionalizing and placement of these sensors in a micro-fluidic channel,
highly
specific and high throughput analysis systems can be realized in a well-
integrated
fashion.
Referring to Fig. 47, exemplary device 900 includes a suspended
structure, for example cantilever 902, coupled to support structure 906 and
having
free end 904 and electrode 908. Electrode 908 is capable of functioning as a
terminal
for DEP. Cantilever 902 may comprise a semiconductor, a metallic conductor, or
a
non-conductor, or any combination thereof. Electrode 908 may be coupled with
or
defined by cantilever 902. For example, electrode 908 may comprise the entire
extent
of or a portion of cantilever 902. For example, electrode 908 may comprise a
thin
metal or degeratively doped semiconductor layer or otherwise defined portion
of
cantilever 902. For example, electrode 908 my include a conductive material
extending to free end 904 of cantilever 902, forming a terminal for
concentrating the
selected analyte at free end 904.
