U.S. patent application number 09/957875 was filed with the patent office on 2002-08-22 for microfabricated ultrasound array for use as resonant sensors.
Invention is credited to Liu, Kelvin J., Nerenberg, Michael I..
Application Number | 20020115198 09/957875 |
Document ID | / |
Family ID | 26927414 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115198 |
Kind Code |
A1 |
Nerenberg, Michael I. ; et
al. |
August 22, 2002 |
Microfabricated ultrasound array for use as resonant sensors
Abstract
Apparatus and methods are provided for microfabricated sensors
for use as resonant sensors. In one embodiment, an array of sensors
is formed by having an electrically common membrane, an insulative
spacer and a base including a driving element. Optionally,
electrostatic drive forces cause the membrane to resonate, and a
binding event is detected. Detection may be capacitive,
piezoelectrical, piezoresistive or optical. Optional vents permit
equilibration to atmosphere. Detection circuitry including phase
lock loop circuitry or tunable oscillator circuitry may be
utilized. High throughput screening, such as for drug discovery can
be achieved.
Inventors: |
Nerenberg, Michael I.; (La
Jolla, CA) ; Liu, Kelvin J.; (San Diego, CA) |
Correspondence
Address: |
FOLEY & LARDNER
402 WEST BROADWAY
23RD FLOOR
SAN DIEGO
CA
92101
|
Family ID: |
26927414 |
Appl. No.: |
09/957875 |
Filed: |
September 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09957875 |
Sep 20, 2001 |
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09845521 |
Apr 26, 2001 |
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60233961 |
Sep 20, 2000 |
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Current U.S.
Class: |
435/287.2 ;
435/287.1 |
Current CPC
Class: |
G01N 2291/106 20130101;
Y10T 29/49126 20150115; G01N 29/02 20130101; G01N 2291/0255
20130101; G01N 2291/0423 20130101; Y10T 29/4913 20150115; B06B
1/0292 20130101; G01N 29/022 20130101; G01N 29/222 20130101; G01N
29/223 20130101; Y10T 29/53052 20150115; G01N 2291/0256 20130101;
G01N 2291/0427 20130101; Y10T 29/49169 20150115; Y10T 29/49128
20150115; G01N 2291/014 20130101; G01N 2291/02818 20130101 |
Class at
Publication: |
435/287.2 ;
435/287.1 |
International
Class: |
C12M 001/34 |
Claims
We claim:
1. A micromechanical sensor comprising a membrane for detecting a
change in the force or membrane surface properties, said sensor
comprising: a substrate; and one or more layers on or in said
substrate, said one or more layers forming a cavity or said
substrate and said one or more layers forming a cavity, said cavity
comprising: one or more side walls; a membrane covering the top of
said cavity, said membrane providing a substantial barrier to
liquid entry through the top of said cavity; and at least two
electrodes, wherein an upper electrode is the membrane or is
fabricated on, within or below the membrane and a lower electrode
below the membrane, wherein said membrane composition and dimension
enables said membrane to vibrate or resonate in response to changes
in electrical signal in said lower electrode, and wherein said
change in force or membrane surface properties are detected by the
sensor as an alteration of the membrane response.
2. The sensor of claim 1, wherein said cavity is substantially
liquid free
3. The sensor of claim 1, wherein said force is pressure.
4. The sensor of claim 1, wherein said membrane surface property
change is an increase in mass associated with the membrane.
5. The sensor of claim 4, wherein said increase in mass results
from a binding event on said membrane.
6. The sensor of claim 5, wherein said binding event is between an
analyte in solution or in a gas and a binding partner immobilized
on the sensor membrane.
7. The sensor of claim 1, wherein said substrate comprises one or
more materials selected from the group consisting of; single
crystal silicon, glass, gallium arsinide, silicon-on-insulator,
silicon-on-sapphire, and indium phosphate.
8. The sensor of claim 1, wherein said one or more layers comprise
one or more materials selected from the group consisting of: single
crystal silicon, polysilicon, silicon nitride, silicon dioxide,
phosphosilicate glass, borophosphosilicate glass, aluminum nitride,
zinc oxide, polyvinylidene fluoride, lead zirconate, and metal.
9. The sensor of claim 1, wherein said one or more layers comprise
materials having different electrical properties.
10. The sensor of claim 1, wherein said substrate comprises a
P-type silicon wafer having a resistance rating between 5 and
15,000.OMEGA..multidot.cm.
11. The sensor of claim 9, wherein said resistance rating is 10,000
.OMEGA..multidot.cm.
12. The sensor of claim 1, wherein said membrane is circular in
shape.
13. The sensor of claim 12, wherein said membrane has a radii of
between 2.5 to 50 microns.
14. The sensor of claim 12, wherein said membrane has a thickness
of between at least 0.05 and 0.5 microns.
15. The sensor of claim 1, wherein said membrane is polygonal in
shape.
16. The sensor of claim 15, wherein the membrane has a length of
between 5 to 100 microns.
17. The sensor of claim 1, wherein said one or more side walls have
a height of between 0.1 to 2 microns.
18. The sensor of claim 1, wherein said membrane comprises one or
more of; single crystal silicon, polysilicon, silicon nitride,
phosphosilicate glass, borosilcate glass, silicon dioxide, aluminum
nitride, zinc oxide, polyvinylidene fluoride, lead zirconate, or
metal.
19. The sensor of claim 1, wherein said cavity has a depth of
between 0.1 to 2 microns.
20. The sensor of claim 1, wherein said cavity has a depth of
between 0.3 to 1 micron.
21. The sensor of claim 1, wherein said cavity comprises one or
more vents connecting said cavity to the exterior of said
sensor.
22. The sensor of claim 1, wherein the cavity comprises one or more
dielectric materials.
23. The sensor of claim 22, wherein said dielectric materials are
selected from the group consisting of; tantalum, polypropylene
film, polymer-aluminum, polyester, metalized polyester, plastic
foam sheet, transformer oils, paraffin, gas, argon, oxygen, and
chlorine.
24. The sensor of claim 1, wherein said cavity comprises an
interior inert ambient atmosphere.
25. The sensor of claim 1, wherein said cavity comprises a
vacuum.
26. The sensor of claim 1, wherein said two or more electrodes
comprise a material selected from the group consisting of; p-doped
silicon, n-doped silicon, metal alloy, titanium, gold, aluminum,
and tungsten.
27. The sensor of claim 1, wherein said upper electrode is the
membrane.
28. The sensor of claim 1 wherein said two or more electrodes
comprises an upper electrode and two lower electrodes, wherein one
lower electrode is a actuation electrode and the other lower
electrode is a detection electrode.
29. The micromechanical sensor of claim 1, wherein said membrane
comprises a binding partner that binds an analyte.
30. The sensor of claim 29, wherein said binding partner is
selected from the group consisting of; antibodies, antigens,
nucleic acid molecules natural DNA, RNA, gDNA, cDNA, MRNA, tRNA,
synthetic DNA, RNA, gDNA, cDNA, mRNA, tRNA, lectins, sugars,
oligosaccharides, glycoproteins, receptors, growth factors,
cytokines, small molecules, peptide library, natural products
library, a legacy library, a combinatorial library, an
oligosaccharide library, a phage display library, metabolites,
drugs of abuse, metabolic by-products of drugs of abuse, enzyme
substrates, enzyme inhibitors, enzyme co-factors, vitamins, lipids,
steroids, metals, oxygen, gases found in physiologic fluids, cells,
cellular constituents, cell membranes, associated cell structures,
cell adhesion molecules, plant products, animal products, and tumor
markers.
31. The sensor of claim 1, wherein said membrane further comprises
one or more piezoresistive elements, wherein said response of said
membrane to said change is force or membrane surface properties is
measured through a change in the resistance of said one or more
piezoresistive elements.
32. The sensor of claim 1, wherein said membrane further comprises
one or more piezoelectric elements capable of producing an output
voltage, and wherein said response of said membrane to said change
is force or membrane surface properties is measured through a
change in output current from said one or more piezoelectric
elements.
33. A sensor array comprising a plurality of micromechanical sensor
sites, said sensor sites comprising a membrane for detecting a
change in force or membrane surface properties, each sensor site
comprising: a substrate; and one or more layers on or in said
substrate, said one or more layers forming a cavity or said
substrate and said one or more layers forming a cavity, said cavity
comprising: one or more side walls; a membrane covering the top of
said cavity, said membrane providing a substantial barrier to
liquid entry through the top of said cavity; and at least two
electrodes, wherein an upper electrode is the membrane or is
attached to the membrane and a lower electrode below the membrane,
wherein said membrane composition and dimension enables said
membrane to vibrate or resonate in response to changes in
electrical current in said lower electrode, and wherein said change
in force or surface membrane properties is detected by the sensor
as an alteration of the membrane response.
34. The sensor array of claim 33, wherein said cavity of each
sensor is substantially liquid free.
35. The sensor array of claim 33, wherein said force is
pressure.
36. The sensor array of claim 33, wherein said membrane surface
change is an increase in mass associated with the membrane.
37. The sensor array of claim 36, wherein said increase in mass
results from a binding event on said membrane.
38. The sensor array of claim 37, wherein said binding event is
between an analyte in solution or in a gas and a binding partner
immobilized on the sensor membrane.
39. The sensor array of claim 33, wherein said substrate comprises
one or more materials selected from the group consisting of; single
crystal silicon, glass, gallium arsinide, silicon insulator,
silicon-on-sapphire, and indium phosphate.
40. The sensor of claim 33, wherein said one or more layers
comprise one or more materials selected from the group consisting
of: single crystal silicon, polysilicon, silicon nitride, silicon
dioxide, phosphosilicate glass, borophosphosilicate glass, aluminum
nitride, zinc oxide, polyvinylidene fluoride, lead zirconate, and
metal.
41. The sensor array of claim 33, wherein said one or more layers
comprise materials having different electrical resistance
properties.
42. The sensor array of claim 33, wherein said substrate comprises
a P-type silicon wafer having a resistance rating between 5 and
15,000.OMEGA..multidot.cm.
43. The sensor array of claim 42, wherein said resistance rating is
10,000 .OMEGA..OMEGA..multidot.cm.
44. The sensor array of claim 33, wherein said membrane is circular
in shape.
45. The sensor array of claim 44, wherein said membrane has a
radius of between 2.5 to 50 microns.
46. The sensor array of claim 33, wherein said membrane has a
thickness between at least 0.05 and 0.5 microns.
47. The sensor array of claim 33, wherein said membrane is
polygonal in shape.
48. The sensor array of claim 33, wherein the membrane has a length
of between 5 to 100 microns.
49. The sensor array f claim 33, wherein said one or more side
walls have a height of between 0.1 to 2 microns.
50. The sensor array of claim 33, wherein said membrane comprises
one or more of; single crystal silicon, polysilicon, silicon
nitride, phosphosilicate glass, borosilcate glass, silicon dioxide,
aluminum nitride, zinc oxide, polyvinylidene fluoride, lead
zirconate, or metal.
51. The sensor array of claim 33, wherein said cavity has a depth
of between 0.1 to 2 microns.
52. The sensor array of claim 33, wherein said cavity has a depth
of between 0.3 to 1 micron.
53. The sensor array of claim 33, wherein said cavity comprises one
or more vents connecting said cavity to the exterior of said
sensor.
54. The sensor array of claim 33, wherein the cavity comprises one
or more dielectric materials.
55. The sensor array of claim 33, wherein said dielectric materials
are selected from the group consisting of; tantalum, polypropylene
film, polymer-aluminum, polyester, metalized polyester, plastic
foam sheet, transformer oils, paraffin, gas, argon, oxygen, and
chlorine.
56. The sensor array of claim 33, wherein said cavity comprises an
interior inert ambient atmosphere.
57. The sensor array of claim 33, wherein said cavity comprises a
vacuum.
58. The sensor array of claim 33, wherein said two or more
electrodes comprise a material selected from the group consisting
of; boron, phosphorus, metal alloy, titanium and tungsten.
59. The sensor array of claim 33, wherein said upper electrode is
the membrane.
60. The sensor array of claim 33 wherein said two or more
electrodes comprises an upper electrode and two lower electrodes,
wherein one lower electrode is a actuation electrode and the other
lower electrode is a detection electrode.
61. The sensor array of claim 33, wherein said membrane comprises a
binding partner that binds an analyte.
62. The sensor array of claim 61, wherein said binding partner is
selected from the group consisting of; antibodies, antigens,
nucleic acid molecules natural DNA, RNA, gDNA, cDNA, mRNA, tRNA,
synthetic DNA, RNA, gDNA, cDNA, mRNA, tRNA, lectins, sugars,
oligosaccharides, glycoproteins, receptors, growth factors,
cytokines, small molecules, peptide library, natural products
library, a legacy library, a combinatorial library, an
oligosaccharide library, a phage display library, metabolites,
drugs of abuse, metabolic by-products of drugs of abuse, enzyme
substrates, enzyme inhibitors, enzyme co-factors, vitamins, lipids,
steroids, metals, oxygen, gases found in physiologic fluids, cells,
cellular constituents, cell membranes, associated cell structures,
cell adhesion molecules, plant products, animal products, and tumor
markers.
63. The sensor array of claim 33, wherein said membrane further
comprises a one or more piezoresistive elements, wherein said
response of said membrane to said change is force or membrane
surface properties is measured through a change in the resistance
of said one or more piezoresistive elements.
64. The sensor array of claim 33, wherein said membrane further
comprises one or more piezoelectric elements capable of producing
an output current, and wherein said response of said membrane to
said change is force or membrane surface properties is measured
through a change in output voltage from said one or more
piezoelectric elements.
65. The sensor array of claim 33, further comprising one or more
reference sensor sites.
66. The sensor array of claim 33, wherein each of said sensor sites
is individually addressable.
67. The sensor array of claim 33, wherein multiple sensor sites are
simultaneously addressable.
68. A method for detecting the presence of an analyte suspected of
being present in a sample, comprising: contacting the sensor of
claim 1 with the sample and detecting a change in the membrane
response, wherein said sensor membrane comprises a binding partner
for the analyte.
69. The method of claim 68, wherein said analyte and binding
partner are selected from the group consisting of; ligand/receptor,
antigen/antibody, enzyme/substrate, DNA/DNA, DNA/RNA, or RNA/RNA,
nucleic acid/protein.
70. The method of claim 69 wherein membrane response is determined
over a period of time.
71. A method for detecting the presence of an analyte suspected of
being present in a sample, comprising: contacting the sensor array
of claim 33 with the sample and detecting a change in the membrane
response of at least one sensor site, wherein said membrane of said
at least one sensor site comprises a binding partner for the
analyte.
72. The method of claim 71 wherein said membrane response is
determined over a period of time.
73. The method of claim 71 wherein said array further comprises one
or more reference sensor sites.
74. A method for determining the rate of binding of a known amount
of analyte in a sample to one more binding partners immobilized on
separate sensor membranes of a sensor array, comprising: contacting
the sensor array of claim 33 with the sample and detecting a change
in the membrane response over a period of time.
75. The method of claim 74, wherein the rate of binding correlates
to the rate constant of reaction between the analyte and binding
partner.
76. A method for determining the rate of binding of an analyte in a
sample to a plurality of binding partners each immobilized on
separate sensor membranes of a sensor array, comprising: contacting
the sensor array of claim 33 with the sample and detecting a change
in the membrane response over a period of time.
77. A method for determining the affinity between an analyte and
binding partner, comprising the steps of: contacting the sensor
array of claim 33 wherein said sensor array comprises one or more
sensor sites each with a membrane comprising said binding partner
with a sample containing a known concentration of said analyte and
detecting a change in the membrane response over a period of time,
removing analyte bound to said sensor and repeating said contacting
and detecting with a sample containing a different concentration of
said analyte, wherein the affinity is determined by comparing the
amount of binding to the concentration of analyte in the sample.
Description
RELATED APPLICATION INFORMATION
[0001] This application is related to U.S. Provisional App. Ser.
No. 60/233,961, entitled "Methods and Apparatus for Synthesis and
Detection of Biological Molecules", filed on Sep. 20, 2000, and a
continuation of U.S. patent application Ser. No. 09/845,521,
entitled "Microfabricated Ultrasound Array For Use As Resonant
Sensors", filed Apr. 26, 2001, both of which are incorporated by
reference herein including any figures and drawings.
FIELD OF THE INVENTION
[0002] The present invention relates to sensors for monitoring a
change in force as applied to a surface membrane or a change in the
surface properties of the sensor membrane. More particularly, the
invention relates to a microfabricated mechanical resonant sensor
that individually or in an array may be used for the
characterization of molecular binding interactions.
BACKGROUND
[0003] This invention relates to the fabrication and use of
acoustic resonant micro-sensors individually or in combination as
an array in screening assays to determine the presence or amount of
an analyte. The sensors of the invention are useful in detecting
analytes in both aqueous and gas environments.
[0004] The following description is provided to assist the
understanding of the reader. None of the information provided or
references cited is admitted to be prior art to the present
invention.
[0005] Technological advances in combinatorial chemistry, genomics,
and proteomics have fostered an increased need for rapid high
throughput (HTP) screening methods able to monitor and/or detect
the reaction between one or more target species and binding
partners or potential binding partners of such targets. Various
systems have been, and are being, explored to detect analytes.
Systems such as affinity chemical sensing, arrayed sensors, and
acoustic sensors are being investigated for their respective
usefulness in detecting analytes in clinical and non-clinical
settings.
[0006] Affinity Chemical Sensing
[0007] Affinity chemical sensing systems attempt to detect
interactions between a target analyte and an appropriate binding
partner. Such systems generally rely on the production or use of a
detectable signal. Affinity chemical sensing systems employ binding
partners which can be discrete molecular species to which the
target analyte specifically binds, or a phase, such as an organic
polymer, into which the target partitions. Covalently attached
labels such as, fluorescent, electrochemical, radioactive, or mass
based-probes are typically employed in such systems. Methods for
determining the presence analytes by using systems that detect the
inherent optical, electrochemical, or physical properties of a
target species or changes in the properties of the layer containing
the binding partner to which a target species binds, have been
employed to detect and/or monitor un-labeled analytes.
[0008] Charych, et al, U.S. Pat. No. 6,022,748, filed Aug. 29,
1997, describe an example of a sensor employing an optically active
sensor coating that changes color upon binding of the target.
Further example of affinity sensing methods are described by W.
Lukosz, "Principles and sensitivities of integrated optical and
surface plasmon sensors for direct affinity sensing and
immunosensing", Biosensors & Bioelectronics 6, 1991, pp.
215-225. Utilization of surface plasmon resonance in sensing
applications is also described by Hanning in U.S. Pat. No.
5,641,640, filed Dec. 29, 1994. A Chemically Selective Field Effect
Transistor (CHEMFET) that determines target binding by monitoring a
signal change on the sensor surface in response to target binding
to the said surface, is described by Shimada in U.S. Pat. No.
4,218,298, filed Nov. 3, 1978. Ribi et al., in U.S. Pat. Nos.
5,427,915 and 5,491,097, filed Aug. 9, 1993 and Feb. 28. 1994
respectively, describe affinity-based microfabricated sensors in
which a measurable change in conductivity of a bio-electric sensor
layer is used to determine binding of a target species.
[0009] Arrayed Sensors
[0010] Arrayed sensors have multiple individually addressable sites
on the device surface which are modified to contain binding
partners for a target molecule to be detected. An example of such a
detection system can be found in U.S. Pat. No. 6,197,503, filed
Nov. 26, 1997 by in Vo-Dinh et al. The patent describes a device
employing multiple optical sensing elements and microelectronics on
a single integrated chip combined with one or more nucleic
acid-based bioreceptors designed to detect optically labeled,
sequence specific genetic constituents in complex samples.
[0011] Other examples of arrayed sensors include: Pinkel et al.,
U.S. Pat. No. 6,146,593 filed Jul. 24, 1997, describe a method for
fabricating biosensors using functionalized optical fibers to
create a high density array of uniquely addressable biological
binding partners; Fodor et al., U.S. Pat. No. 6,124,102 filed Apr.
21, 1998 describe an optical sensor array having a planar surface
derivatized with ligands of an optically active target species
immobilized at known locations such that each location comprises a
"pixel" of an optical read out device. These and similar devices
can be successful for arrayed detection and therefore useful for
parallel screening of multiple interactions where the analyte is
either labeled or inherently optically, electrically, or
specifically chemically active.
[0012] Acoustic Sensors
[0013] Another field of technology having combine arrayed sensors
is that of sensors based on bulk or microfabricated resonant
devices. Such sensors have been demonstrated in systems used to
determine 3-dimensional acceleration, speed, and position, as
transducers for monitoring environmental conditions such as
pressure, fluid flow, temperature, and as gravimetrically sensitive
elements in chemical affinity sensors.
[0014] Acoustic sensors for chemical sensing have been demonstrated
in low-density arrays in for example Ballato U.S. Pat. No.
4,596,697 filed Sep. 4, 1984 which describes surface acoustic wave
(SAW) devices. Arrays of cantilever sensors for gas phase sensing
of multiple analytes are described by Lang et al (Lang, H. P.;
Baller, M. K.; Berger, R.; Gerber, Ch.; Gimzewski, J. K.;
Battiston, F. M.; Fomano, P.; Ramseyer, J. P.; Meyer, E.;
Guntherodt, H. J.; IBM Research Report, RZ 3068 (#93114), Oct. 19,
1998), and Britton et al (Britton, C. L.; Jones, R. L.; Oden, P.
I.; Hu, Z.; Warmack, R. J.; Smith, S. F.; Bryan, W. L.; Rochelle,
J. M.; Ultramicroscopy, 82, 2000, p. 17-21).
SUMMARY OF THE INVENTION
[0015] The invention described herein relates to microfabricated
resonant sensors that can be used individually or as an
interconnected yet electrically isolated grouping in microarrays.
The invention relates to an electromechanical sensor for monitoring
a change in surface properties of a sensor membrane. The change in
surface properties results from a binding event that changes the
physical characteristics of the membrane surface, such as surface
mass, viscous coupling, membrane stiffness, and the like. The
sensors of the invention can also be used to determine a change in
force on the surface of a sensor membrane, such as results from a
binding event or application of pressure. A sensor can be part of
an array of sensors which can be fabricated to high density. The
sensors of the invention have many applications including, for
example, to determine the presence or amount of an analyte in a
sample from a clinical, research or natural environment. In this
case, a binding partner of the analyte can be immobilized to the
resonant sensor membrane surface and the binding of analyte to the
binding partner on the membrane can be identified through a shift
in the resonant characteristics of the sensor membrane.
[0016] The term "sensor" as used herein relates to an apparatus or
device that can respond to an external stimulus such as, a change
in mass on a surface, pressure, force, or a particular motion,
where the apparatus can transmit a resulting signal to be measured
and/or detected.
[0017] The term "binding event" refers to an interaction or
association between a minimum of two molecular structures, such as
an analyte and a binding partner. The interaction may occur when
the two molecular structures are in direct or indirect physical
contact. Examples of binding events of interest in the present
context include, but are not limited to, ligand/receptor,
antigen/antibody, enzyme/substrate, DNA/DNA, DNA/RNA, RNA/RNA,
nucleic acid mismatches, complementary nucleic acids, nucleic
acid/proteins, and the like.
[0018] As used herein "microfabricated" refers to the procedures
and/or methods, such as bulk and surface micromachining, used to
etch, deposit, pattern, dope, form and/or fabricate structures
using substrates such as silicon and the like. Microfabrication
procedures are known in the art and have been used to prepare
microsystems such as computer processor chips, acoustic sensors,
micro-circuits and other devices requiring micron and nanomolecular
scale portions used in fields such as microengineering.
[0019] In one aspect, the present invention provides a
micromechanical sensor for detecting a change in force at a
membrane surface or a change in the surface properties of the
sensor membrane. The apparatus or sensor of the invention comprises
a substrate and one or more layers formed on or in the substrate.
The substrate and/or layers form a cavity comprising one or more
side walls and a membrane that covers the top of the cavity. The
cavity side walls can be flat, angled, sloped or curved. In
preferred embodiments, the membrane provides a substantial barrier
to liquid entry through the top of the cavity. The cavity also
comprises at least two electrodes, which include an upper electrode
and a lower electrode. The upper electrode can be the membrane
itself or the upper electrode can be fabricated on, within or below
the membrane. The lower electrode is below the membrane. The
composition and dimension of the membrane are chosen so that it can
vibrate or resonate in response to changes in electrical signal
from the lower and/or upper electrode. Preferably, the membrane is
responsive to a change in resonant frequency and/or a harmonically
varied electrical current. More preferably, the membrane is
harmonically responsive to a change in force on the membrane
surface or surface properties of the membrane, for example a
binding event near and/or on the membrane surface. Preferably the
diameter or width of a sensor of the invention is between at least
5 and up to 200 microns. More preferably the diameter or width of a
sensor is between 10 to 100 microns.
[0020] "Liquid free" as used herein refers to the micromachined or
naturally occurring cavity of the sensor being substantially free
of any fluid or fluid-like material, for example water or
gelatinous materials.
[0021] The term "analyte" or "target" refers to any molecule being
detected by the sensor. The analyte (or target) is detected by
immobilizing one or more binding partners (or "probes") or presumed
binding partners specific for the analyte or target to a sensor
membrane. Thus, when it is desired to use the sensor to determine
if a gas or solution contains an analyte, the surface of the sensor
membrane that is to contact the gas or solution is immobilized with
a binding partner for that analyte. Analyte and its binding partner
represent a binding pair of molecules, which interact with each
other through any of a variety of molecular forces including, for
example, ionic, covalent, hydrophobic, van der waals, and hydrogen
bonding, so that the pair have the property of binding specifically
to each other. Specific binding means that the binding pair exhibit
binding with each other under conditions where they do not bind to
another molecule. Examples of types of specific binding pairs are
antigen-antibody, biotin-avidin, hormone-receptor, receptor-ligand,
enzyme-substrate, lgG-protein A, and the like.
[0022] Preferred binding partners and/or analytes of the present
invention include, but are not limited to, antibodies, antigens,
nucleic acids (e.g. natural or synthetic DNA, RNA, gDNA, cDNA,
mRNA, tRNA, etc.), lectins, sugars, oligosaccharides,
glycoproteins, receptors, growth factors, cytokines, small
molecules such as drug candidates (from, for example, a random
peptide library, a natural products library, a legacy library, a
combinatorial library, an oligosaccharide library and a phage
display library), metabolites, drugs of abuse and their metabolic
by-products, enzyme substrates, enzyme inhibitors, enzyme
co-factors such as vitamins, lipids, steroids, metals, oxygen and
other gases found in physiologic fluids, cells, cellular
constituents, cell membranes and associated structures, cell
adhesion molecules, natural products found in plant and animal
sources, tumor markers (i.e., molecules associated with tumors),
other partially or completely synthetic products, and the like.
[0023] Analytes or binding partners bay be naturally occurring or
synthetically prepared. A "natural analyte" is an analyte which
occurs in nature and specifically binds to a particular site(s) on
a particular binding partner such as a protein. Examples by way of
illustration and not limitation include a receptor and a ligand
specific for the receptor (e.g., an agonist or antagonist), an
enzyme and an inhibitor, substrate or cofactor; and an antibody and
an antigen.
[0024] The terms "isolated," "purified," or "biologically pure"
mean an object species is the predominant species present (i.e., on
a molar basis it is more abundant than any other individual species
in the composition), and preferably a substantially purified
fraction in a composition wherein the object species comprises at
least about 50 percent (on a molar basis) of all macromolecular
species present. Generally, a substantially pure composition will
comprise more than about 80 to 90 percent of all macromolecular
species present in the composition. Most preferably, the object
species is purified to essential homogeneity (contaminant species
cannot be detected in the composition by conventional detection
methods) wherein the composition consists essentially of a single
macromolecular species.
[0025] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and also encompasses known analogs of natural nucleotides that can
function in a similar manner as naturally occurring
nucleotides.
[0026] "Polypeptide", "peptide," "protein" and "protein target" are
used interchangeably to refer to a polymer of amino acid residues.
The terms apply to amino acid polymers in which one or more amino
acid residue is an artificial chemical analogue of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. An analyte and/or its binding partner can be a
protein. The protein or protein target to which ligands are being
screened in drug discovery methods is essentially any type capable
of binding some type of ligand including, by way of example and not
limitation, for example, enzymes, receptors, antibodies and
fragments thereof, hormones, and nucleic acid binding proteins. A
protein or peptide may include a particular site, this site is the
site at which a ligand and the protein or peptide form a binding
complex. For an enzyme, the particular site can be the active site
or an allosteric site; in the instance of a receptor, the
particular site is the site at which a natural ligand binds.
[0027] The term "antibody" refers to a protein consisting of one or
more polypeptides substantially encoded by immunoglobulin genes or
fragments of immunoglobulin genes. The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon and
mu constant region genes, as well as myriad immunoglobulin variable
region genes. Light chains are classified as either kappa or
lambda. Heavy chains are classified as gamma, mu, alpha, delta, or
epsilon, which in turn define the immunoglobulin classes, IgG, IgM,
IgA, IgD and IgE, respectively.
[0028] A typical immunoglobulin (antibody) structural unit is known
to comprise a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (VL) and variable heavy chain (VH) refer to
these light and heavy chains respectively. An antibody can be
specific for a particular antigen. The antibody or its antigen can
be either an analyte or a binding partner.
[0029] Antibodies exist as intact immunoglobulins or as a number of
well-characterized fragments produced by digestion with various
peptidases. Thus, for example, pepsin digests an antibody below the
disulfide linkages in the hinge region to produce F(ab)'.sub.2, a
dimer of Fab which itself is a light chain joined to VH-CH1 by a
disulfide bond. The F(ab)'.sub.2 may be reduced under mild
conditions to break the disulfide linkage in the hinge region
thereby converting the (Fab').sub.2 dimer into an Fab' monomer. The
Fab' monomer is essentially an Fab with part of the hinge region
(see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y.
(1993), for a more detailed description of other antibody
fragments). While various antibody fragments are defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that such Fab' fragments may be synthesized de novo
either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein also includes antibody
fragments either produced by the modification of whole antibodies
or synthesized de novo using recombinant DNA methodologies.
Preferred antibodies include single chain antibodies, more
preferably single chain Fv (scFv) antibodies in which a variable
heavy and a variable light chain are joined together (directly or
through a peptide linker) to form a continuous polypeptide.
[0030] A single chain Fv ("scFv") polypeptide is a covalently
linked VH::VL heterodimer which may be expressed from a nucleic
acid including VH- and VL-encoding sequences either joined directly
or joined by a peptide-encoding linker. Huston, et al. (1988) Proc.
Nat. Acad. Sci. USA, 85:5879-5883. A number of structures for
converting the naturally aggregated--but chemically separated light
and heavy polypeptide chains from an antibody V region into an scFv
molecule which will fold into a three dimensional structure
substantially similar to the structure of an antigen-binding site.
See, e.g. U.S. Pat. Nos. 5,091,513 and 5,132,405 and 4,956,778.
[0031] An "antigen-binding site" or "binding portion" refers to the
part of an immunoglobulin molecule that participates in antigen
binding. The antigen binding site is formed by amino acid residues
of the N-terminal variable ("V") regions of the heavy ("H") and
light ("L") chains. Three highly divergent stretches within the V
regions of the heavy and light chains are referred to as
"hypervariable regions" which are interposed between more conserved
flanking stretches known as "framework regions" or "FRs". Thus, the
term "FR" refers to amino acid sequences that are naturally found
between and adjacent to hypervariable regions in immunoglobulins.
In an antibody molecule, the three hypervariable regions of a light
chain and the three hypervariable regions of a heavy chain are
disposed relative to each other in three dimensional space to form
an antigen binding "surface". This surface mediates recognition and
binding of the target antigen. The three hypervariable regions of
each of the heavy and light chains are referred to as
"complimentarily determining regions" or "CDRs" and are
characterized, for example by Kabat et al. Sequences of proteins of
immunological interest, 4th ed. U.S. Dept. Health and Human
Services, Public Health Services, Bethesda, Md. (1987). An epitope
is that portion of an antigen that interacts with an antibody.
[0032] "Sample" refers to essentially any source from which an
analyte can be obtained. A sample may be acquired from essentially
any organism, including animals and plants, as well as cell
cultures, recombinant cells, cell components and can also be
acquired from environmental sources. Samples can be from a
biological tissue, fluid or specimen and may be obtained from a
diseased or healthy organism. Samples may include, but are not
limited to, sputum, amniotic fluid, blood, blood cells (e.g., white
cells), urine, semen, peritoneal fluid, pleural fluid, tissue or
fine needle biopsy samples, and tissue homogenates. Samples may
also include sections of tissues such as frozen sections taken for
histological purposes. Typically, samples are taken from a human.
However, samples can be obtained from other mammals also, including
by way of example and not limitation, dogs, cats, sheep, cattle,
and pigs. The sample may be pretreated as necessary by dilution in
an appropriate buffer solution or concentrated, if desired. Any of
a number of standard aqueous buffer solutions, employing one of a
variety of buffers, such as phosphate, Tris, or the like,
preferably at physiological pH can be used. A sample also my be
artificially prepared such as a control sample that contains a
known amount of an analyte.
[0033] By "environmental sources" it is meant potentially any place
in the natural and/or man-made environment from which a sample can
be taken. Environmental sources include: water sources such as
oceans, lakes, ponds, rivers and streams; earthen sources such as
soil, sand, interior or exterior dust; gas sources such as air,
such as polluted and/or non-polluted air from our general
surroundings or from industrial plants or automotive exhaust and
the like.
[0034] Biological samples can be derived from patients using well
known techniques such as venipuncture, lumbar puncture, fluid
sample such as saliva or urine, or tissue biopsy and the like.
Biological samples also include exhaled air samples as taken with a
breathalyzer or from a cough or sneeze. A biological sample may be
obtained from a cell or blood bank where tissue and/or blood are
stored, or from an in vitro source, such as a culture of cells.
Techniques for establishing a culture of cells for use as a source
for biological materials are well known to those of skill in the
art. Freshney, Culture of Animal Cells, a Manual of Basic
Technique, Third Edition, Wiley-Liss, N.Y. (1994) provides a
general introduction to cell culture.
[0035] As used herein the term "membrane response" relates to the
vibration or resonance of the membrane layer that is extended over,
or placed on, and roughly covers, in a sealed liquid impermeable,
manner a cavity of the invention sensor. Upon the introduction of a
current or formation of an electrostatic potential, the membrane of
the invention can move, vibrate or oscillate in a manner that can
be measured, for example, acoustically, electronically by
electromechanical transduction such as by
electrostatics/capacitance, piezoresistance or piezoelectricity, or
optically by interferometry, such as laser-Doppler vibrometery. The
extent of vibration or oscillation of the membrane depends, for
example, on the physical properties of the membrane and its
relation to another electrode in the cavity or the effect of mass
or force on the membrane surface.
[0036] The term "substrate" is used herein to refer to the starting
material from which the sensor of the invention is fabricated. The
substrate can comprise single crystal silicon, glass, gallium
arsinide, silicon insulator, silicon-on-sapphire, and indium
phosphate, and the like. Also, combinations of these materials can
be used. Preferably, the substrate has a high electrical
resistance, such as a P or N-type silicon wafer rated up to
15,000.OMEGA..multidot.cm. In a particular embodiment, the
substrate comprises a silicon wafer, double side polished, P or
N-type substrate having a resistance between 5 and 1
5,000.OMEGA..multidot.cm. More preferably, the substrate is a
double side polished, silicon wafer, P or N-type having a
resistance of roughly 10,000.OMEGA..multidot.cm. The substrate of
the sensor can comprises one or more dopants, for example boron
and/or phosphorus, to be patterned as one or more electrodes, and
any vents, passages or holes within the cavity can extend through
the substrate.
[0037] Sensor layers that are added to the substrate during sensor
fabrication can comprise single crystal silicon, polysilicon,
silicon nitride, silicon dioxide, phosphosilicate glass,
borophosphosilicate glass, aluminum nitride, zinc oxide,
polyvinylidene fluoride, lead zirconate, metal, and the like.
Combinations of these materials also can be used. The layers can
have different electrical properties from the substrate. For
example, some layers of a sensor can be used to aid in electrically
isolating one region, the upper region for example, of a sensor
from a lower region, or in another example, can provide electrical
isolation of the membrane and the electrode(s). Layers also may be
used to form electrodes and/or electrode leads. For example, a
sensor layer having a cavity, can have a via, or a channel etched
in the most planar surface, the XY horizontal surface of the
substrate, connecting an electrode to a side wall, or the base of
the cavity. This channel can be lined with a passivating layer and
filled with a doped polysilicon, or a metal, such as titanium,
metal alloy, titanium, gold, platinum, tungsten, aluminum, and the
like, and then an additional layer having a different resistance
can be placed on top of the now filled channel. In forming such
layers, and in preparing electrodes and leads of the present
invention it would be recognized that the area of the sensor that
carries an electrical charge from an electrical power source should
be electrically isolated from other regions of the sensor in order
to avoid a failure of conductivity, or a short, of the electrodes
and leads.
[0038] In another embodiment, the sensor layers and electrodes can
be arranged by taking a substrate and applying a passivating layer,
for example oxide or nitride, to provide an insulating layer
between the substrate and any electrodes. Electrodes can be formed
by depositing and patterning metal on the surface of the
passivating layer. Another layer, a patterned spacer layer can be
added with an area of the spacer layer being defined as the sensor
cavity. The cavity of the sensor can be formed by etching away the
defined area in the spacer layer and preferably the cavity is
formed above the electrode. A membrane layer is then placed over
and sealed on top of the cavity.
[0039] As used herein, "electrically isolate," "electrical
isolation," and like terms when used in reference to electrodes,
leads, arrays and sensors of the invention, refer to arranging
sensors and components of sensors in a manner that insulates the
array, sensor, electrode or lead that transports or carries a
current of electricity from surrounding layers, sensors, electrodes
or leads. For example an array having more than one sensor in close
proximity to other sensors that are electrically isolated can have
essentially all of a current applied to at least one sensor in the
array. Electrical isolation of sensor elements in an array can be
accomplished by forming a p/n-junction between the conducting paths
and the substrate. A p-n junction can be formed by doping the two
halves of a single piece of a semiconductor, or two opposing
layers, so that they become, respectively, p-type and n-type
material, by doing this an interface is formed between the two
halves creating a p-n junction. Such p-n junctions have the
property that it does not allow current will flow and the junction
is said to be backward biased. In another embodiment, the sensor,
array, lead or electrode can be insulated by ensuring that
materials used to surround the component are incapable of
conducting an electrical current. Electrical isolation could also
be obtained by physically separating sensors in an array, by
arranging the sensors in a manner in which they do not
substantially contact another sensor yet are present on the same
array.
[0040] The membrane of the sensor can be polygonal or elliptical.
In a preferred embodiment, is rectangular or square having sides of
5 to 100 microns in length. More preferably, the membrane is
circular having a radius of 2 and up to 100 microns, 2 to 30 or 2.5
to 50 microns in length. The membrane of the invention covers a
prepared, micromachined, microfabricated or naturally occurring
cavity in the substrate and can cover the cavity in a manner that
prevents a liquid from entering the cavity. Preferably the membrane
is up to 0.5 microns thick. More preferably the membrane is at
least 0.05 and up to 0.5 microns thick.
[0041] The membrane or membrane layer of the sensor can be
fabricated from an electrically conductive material, such as doped
single crystal silicon, doped polysilicon, metal or any composite
thereof, and can serve as a connection to ground. In alternative
embodiments, the membrane can be fabricated out of non-conductive
materials such as silicon nitride, silicon dioxide, phosphosilicate
glass, borophosphosilicate glass. In this case, the membrane is not
an electrode but can have an electrode fabricated within, on, above
or below the surface. As discussed herein, the membrane covers
roughly the entire opening of the cavity in a substantially sealed
manner. The membrane of the sensor can also serve to conduct an
electrical signal. In another embodiment the membrane layer can be
fabricated to contain one or more secondary structures that can
conduct a current of electricity such as piezoelectric or
piezoresistive materials. In selecting a material to serve as a
membrane for the invention sensor, certain mechanical
characteristics such as Young's Modulus, which refers to the
stiffness of the membrane, the density, the intrinsic stress, and
internal damping are considered. In a preferred embodiment of the
present invention the membrane is prepared or fabricated in a
manner that allows the membrane to vibrate and/or resonate. The
membrane can also be fabricated to either serve as an electrode for
conducting electricity, or as a connection to ground. The membrane
can serve as part of a capacitive or electrostatic pair. Within
this embodiment, the membrane and the other electrode of the pair
are separated by the space of the cavity and/or materials within
the cavity, and act as a capacitor like structure.
[0042] In another embodiment the present invention provides a
sensor comprising a cavity that is preferably 0.1 to 2 microns deep
and sealed with an addressable, conductive membrane. In a preferred
embodiment the membrane is preferably 0.1 to 0.5 microns in
thickness. The sensor membrane resonate or vibrate and can be used
as a chemical affinity sensor. For example, the topmost surface of
the membrane can be derivatized to comprise at least one member of
a binding pair. In following, upon exposure to a solution or gas
phase that contains a second member of the binding pair, binding
occurs between the two molecules and an increase in mass relative
to the mass of the single membrane bound member occurs on the
membrane. This change in mass at the surface of the membrane can
alter the resonant characteristics of the membrane and/or the
fundamental frequency of vibration or the phase of a vibration
relative to a driving signal of the membrane can be said to change
and/or shift.
[0043] By the term "addressable" when describing the electrical
potential of the sensor, membrane, electrode or an array, is meant
that the described layer, sensor, substrate and/or membrane can
accept an electron, have an electric potential or voltage
assignment. The electric potential can be the assignment of having
a ground voltage, such as for example the membrane can be held at
ground voltage when a sensor operates using an AC, alternating
current, power source, or the assignment can be a lower or higher
electric potential within the membrane in reference to an opposing
electrode if using a DC, direct current, electrode power source.
The term "addressable" when used to describe a sensor when placed
in an array, combines the concept of assigning an electric
potential or voltage and relates to each sensor being capable of
being given a specific locator and/or identifier, allowing a
particular sensor in an array to be separately identifiable from
surrounding sensors when used in methods such as high through put
screening.
[0044] As discussed above, the present invention sensor comprises a
cavity that is covered by a membrane as described herein.
Preferably, the cavity comprises one or more walls that are 0.1 to
2 microns in height. More preferably, the cavity walls are 0.3 to 1
micron in height, creating a cavity or a well that is roughly 0.3
to 1 micron deep. It is the distance between the upper electrode
and lower electrode that determines the height of the cavity. Thus,
the distance from the lower to upper electrode is roughly 0.3 to 1.
In a preferred embodiment, the cavity is completely covered in a
sealed, liquid free manner by the membrane. In another embodiment,
the membrane that completely covers the cavity comprises one or
more holes on its surface.
[0045] Within an aspect of the present invention, it is the cavity
that serves as the dielectric space between the electrode in the
bottom of the cavity and the upper electrode. It would be
understood by one of ordinary skill in the art that combination of
the lower and upper electrode comprise a capacitor system where the
cavity is, as stated, the dielectric space through which an
electrostatic field can be formed.
[0046] In another embodiment, the cavity of the sensor has
passages, or vents, which are holes in the substrate that connect
the exterior of the sensor to the cavity. In a preferred
embodiment, the cavity can be vented, having holes, tunnels or
pores which allow the cavity to be vented to the external
atmosphere of the sensor. Vents in the cavity of the sensor
function to eliminate and/or relieve the effects of barometric
pressure variation and pressurization in the cavity during
operation of the device. The passages or vents can be in the base,
the sidewalls of the cavity, or in the membrane which covers the
cavity. Thus, in a particular aspect, the sensor of the invention
has a sealed cavity being covered by a membrane having vents. The
cavity can be partially filled, or filled with a dielectric or
multiple dielectric materials or gases, such as tantalum,
polypropylene film, polymer-aluminum, polyester, metalized
polyester, plastic foam sheet, transformer oils such as paraffin,
gasses such as argon, oxygen, chlorine, and any mixture of the
like. Preferably, the cavity comprises at least one passage or vent
which passes in the Z or perpendicular dimension, through the
substrate and to the exterior and/or ambient surroundings of the
sensor. The vents and/or passages can extend in a horizontal or
planar manner from the cavity through the walls of the cavity well
or in another embodiment the vents and/or passages can extend
through the floor of the cavity leading in a perpendicular manner
to the exterior of the sensor.
[0047] The sensor of the present invention can comprise at least
two electrodes as mentioned above. Electrodes in the sensor cavity
are preferably planar. The electrodes of the sensor can be created
by etching vias or channels into the substrate, providing a
passivating insulating layer and implanting or sputtering a metal,
for example titanium, gold, platinum, tungsten, a metal alloy
and/or other like metals. It is also an aspect of the invention to
dope the substrate with an impurity which depending on the type of
substrate chosen, p-type or n-type, can indicate either a substance
such as boron, P-type, or phosphorus, N-type, to act as leads
and/or electrodes. In another embodiment, the electrodes of the
sensor can be formed of one or more metal or diffused dopant
electrode layers in the bottom of the cavity.
[0048] Leads are used to connect electrodes to a power source or
ground. The leads can be prepared by fabricating holes or tunnels
through the substrate cavity which lead away from the cavity in a
perpendicular manner. Such perpendicular leads can be prepared to
extend through the substrate to the exterior of the sensor in order
to be connected to an electrical current source, or the leads can
extend from the substrate cavity floor and be configured to exit
the sensor at an angle, through one or more sides of the sensor
itself.
[0049] In another embodiment, the electrodes can be part of, or
reside on or within the membrane of the sensor. One of ordinary
skill in the art would recognize that preparation of electrodes on,
within or under the membrane should not interfere with either the
acoustics of the cavity, nor should they interfere with the
resonance, oscillation or vibrations of the membrane itself. It is
within this aspect of the invention that one or more electrodes are
prepared on, within or under the membrane to serve as actuating
electrodes and/or sensing electrodes, and it is also within aspects
of the invention to have electrodes on, within or under the
membrane and have additional electrodes as described above.
[0050] Resonation or vibration of the membrane can be initiated
electrostatically through use of electrodes in the sensor base, the
membrane, the cavity wall, the cavity floor and/or membrane where
the electrodes are connected in a manner that allows the initiation
or creation of an electric current and/or potential. Resonation or
vibration of the membrane of the sensor can be monitored using
electrodes that can be located in and around the sensor as
described and illustrated herein, and which can be part of a
monitor apparatus, or monitoring can occur, for example, either
acoustically, electronically by electromechanical transduction such
as by electrostatics/capacitance, piezoresistance or
piezoelectricity, or optically by interferometry, such as
laser-Doppler vibrometery.
[0051] In another aspect of the present invention, the above
described sensor(s) can be arranged in an array from as few as a
handful of sensor sites to as many as 500,000 individual sensors
per cm.sup.2. High density arrays can comprises between 256 to
150,000 individual sensors/cm.sup.2, and more preferably between
5,000 to 100,000 sensors/cm.sup.2. Each sensor in the array can be
fabricated to generally function similarly. However, in some
embodiments, individual sensor sites may have different types of
sensors, which differ in their mode of operation. It is preferred
that individual sensors sites are arranged in the array in a manner
that allows for electrical isolation of each sensor. In some
embodiments, the individual sensor sites can be individually
addressed. In other embodiments, multiple sensor sites may be
linked so that they can be actuated and detected
simultaneously.
[0052] In another embodiment, the present invention provides high
density arrays having multiple sensors where individual sensors of
the array have a cavity differing in width, depth and shape. The
individual sensors of an array can also comprise membranes of
different width and composition. For example, one or more sensors
of an array can comprise membranes with or without holes on their
surfaces. In a preferred embodiment, the individual sensors of high
density arrays comprise the same cavity shape and depth and further
comprise membranes and substrates of the same width and
composition. More preferably, the individual sensors of an array
are essentially identical in shape and composition, and are
individually addressable. Within this aspect of the invention a
high density array can also comprise sensors that are grouped
together to detect the same analyte.
[0053] Arrays of the present invention can be used in multi-plexed
assays which can be considered assays where more than one analyte
is detected in a sample. For example an array can be prepared with
multiple sensors each having different binding partners. A sample
believed to contain any of the multiple analytes of interest can be
placed in contact with the sensor array and various individual
sensors can be monitored, based on the membrane response, to
determine which analytes are present in the sample. It would be
understood by those of skill in the art that more than one sensor
site in the array can be used as a control or reference sensor to
determine reference values for the assay, such as the baseline
response for the membranes. It would also be recognized by those
skilled in the art that due to the size of the arrays, multiple
arrays with probes for multiple targets can be used
simultaneously.
[0054] In another aspect of the present invention, a
micromechanical sensor for the detection of a change in mass at a
membrane surface of the sensor is provided. Preferably the change
in mass is directly related to a binding event on or near the
surface of the membrane. As discussed above, the binding event can
be between biological or chemical molecules and can be obtained
from a variety of samples. Within this aspect of the invention,
various assays that rely on the binding of one molecule to another
can be utilized. Fields such as immunology, pharmacology, biology,
medicine, chemistry, molecular biology and other like fields of
science have long utilized assays involving molecular and chemical
binding events. Such assays including ELISA, DNA hybridization,
immunoassay, competitive binding assays, sensitivity assays,
affinity and rate binding assays and the like, rely on detection of
optically or chemically detectable marker such as fluorescent
marker which is bound to the analyte of a binding pair. The present
sensor or sensor arrays can perform these same assays and use the
same marker labeled reagents, but in this case, the labeled
reagents are detected though their increased mass, which is
detected due to sensor related detectable changes in membrane
resonance or vibration. Thus, while the invention described herein
has been discussed in terms of comprising sensors and arrays that
can be used with our without detectable labels, it is important to
understand that the term `detectable labels` refers to labels as
normally used in the art to detect a bound analyte through the use
of chemical, radioactive and/or optical means. However, the present
invention can use detectable labels which relate to aspects of the
present invention. With regard to the present invention, a
detectable label can be a molecule or substance that is attached to
a binding partner for an analyte of interest, or a binding partner
which binds to an analyte of interest that adds a certain amount of
additional mass to make the detection for readily detected. Thus, a
detectable label of the invention can be a label that adds a
particular amount of additional molecular mass to a bound pair, or
deposits, upon enzymatic reaction, a detectable amount of molecular
material to the surface of the substrate in response to a probe or
a target that is bound thereon.
[0055] Sensor or sensor arrays of the present invention also can be
used to determine known or unknown analytes in a sample using
direct and indirect binding, competitive inhibition, sensitivity
testing, specificity testing, affinity determination, and the like.
For example, indirect binding may be used when the amount of
analyte that binds to the sensor membrane surface is too low for
the sensor to detect. In this case, the sensor can be contacted
with a sample containing a binding partner specific for the analyte
bound to the sensor membrane. The sample-containing binding partner
is preferably specific for site on the analyte that is separate and
non overlapping from the site bound by the membrane immobilized
binding partner such that the two binding partners can be bound
simultaneously to a single analyte molecule. Thus, indirect
detection is achieved when the additional mass attributed to
binding of the sample-containing binding partner to analyte on the
membrane becomes detectable. Competitive inhibition may be used
with a sensor or sensor array of the invention when an inhibitor
analyte of lower mass inhibits binding of a larger mass analyte to
the membrane.
[0056] In another aspect of the present invention, a method is
provided for determining the presence or amount of an analyte in a
sample. Exemplary steps of the method comprise; acquiring a sample
presumed to contain an analyte to be detected, contacting the
sample with a sensor or sensor array having a membrane that has a
binding partner for the analyte immobilized on the surface, and
determining the presence of the analyte in the sample based on a
measured detectable change in membrane resonance or vibration. In a
preferred embodiment the analyte to be detected and/or the binding
partner of the analyte are not labeled and the sample is a liquid
or gas.
[0057] In another aspect, the present invention provides a method
for determining the rate of binding of a known amount of analyte in
a sample to one more binding partners immobilized on separate
sensor membranes of a sensor array. The method comprises contacting
a sensor array of the invention with the sample and detecting a
change in the membrane response over a period of time. In preferred
embodiments, the rate of binding which occurs over time correlates
to the rate constant of reaction between the analyte and its
binding partner
[0058] In another aspect, the present invention provides a method
for determining the affinity between an analyte and its binding
partner, comprising contacting a sample containing a known
concentration of the analyte with a sensor array of the invention
wherein the sensor array comprises one or more sensor sites each
with a membrane comprising a binding partner for the analyte. A
change in the membrane response over a period of time is then
detected. The bound analyte is then removed and the step of
contacting and detecting with a sample repeated with a different
concentration of the analyte. This cycle of removal, contacting
with a different analyte concentration and measuring membrane
response over time may be repeated multiple times, each with a
different analyte concentration. The binding affinity between the
analyte and its binding partner can be derived by relating binding
rate to analyte concentration in a manner well known to those of
skill in the art.
[0059] While aspects and embodiments of the present invention are
described herein, it would be understood that such descriptions are
exemplary of uses and aspects of the presently described sensors
and arrays and should not limiting in content.
DESCRIPTION OF DRAWINGS
[0060] FIG. 1a is perspective view of an embodiment of individual
resonant micromechanical membrane sensor based on electrostatic
capacitance.
[0061] FIG. 1b is a vertical cross-section of the sensor of FIG. 1
taken substantially along the line 1b-1b of FIG. 1.
[0062] FIG. 2 is a vertical cross-section of an embodiment of an
individual resonant micromechanical membrane sensor based on
electrostatic capacitance.
[0063] FIG. 3 is a vertical cross-section of an embodiment of an
individual resonant micromechanical membrane sensor based on
electrostatic capacitance.
[0064] FIG. 4 is a perspective view of the lower portion of an
embodiment of a resonant micromechanical membrane sensor showing
discrete concentric and sense electrodes ("dual port").
[0065] FIG. 5a is a perspective view of an individual resonant
micromechanical membrane sensor with electrostatic drive and
piezoresistive sense.
[0066] FIG. 5b is a cross-section of the sensor of FIG. 5a, taken
substantially along the lines of 5b-5b.
[0067] FIG. 6a is a perspective view of an individual resonant
micromechanical membrane sensor with electrostatic drive and
piezoelectric sense.
[0068] FIG. 6b is a cross-section of the sensor of FIG. 6a, taken
substantially along the lines of 6b-6b.
[0069] FIG. 7 is a cross-section of an individual resonant
micromechanical membrane sensor showing placement of a vertical
lead from the planar electrode to the outside of the sensor.
[0070] FIG. 8 is a cross-section of an individual resonant
micromechanical membrane sensor showing placement of a horizontal
lead from the planar electrode to the outside of the sensor.
[0071] FIG. 9a is plan view of a resonant micromechanical membrane
sensor array showing membrane, spacer layer and drive elements.
[0072] FIG. 9b is the underside of the resonant micromechanical
membrane sensor array shown in FIG. 9a.
[0073] FIG. 10a is a graph of membrane sensitivity vs. thickness
for 10-micron membrane in air.
[0074] FIG. 10b is a graph of membrane sensitivity vs. thickness
for 10-micron membrane in water.
[0075] FIG. 11a is a graph of resonant membrane frequency in air as
a function of membrane thickness and radius.
[0076] FIG. 11b is a graph of resonant membrane frequency in water
as a function of membrane thickness and radius.
[0077] FIG. 12A-E depicts an approach for fabricating an individual
micromachined resonant membrane sensor.
[0078] FIG. 13 is a schematic of a white noise/fft (Fast Fourier
Transform) scheme.
[0079] FIG. 14 is a schematic of a phase locked loop.
[0080] FIG. 15a is a diagram of probes bound to an array.
[0081] FIG. 15b is a diagram of probes bound to single membrane of
an individual micromachined resonant membrane sensor.
DETAILED DESCRIPTION
[0082] Individual Sensor Embodiments
[0083] The present invention provides a resonant micromechanical
membrane sensor in both single and array formats that is sensitive
to changes in the surface properties of the membrane surface such
as density, inertia, viscous drag, or force. Measurement of a mass
change using the sensors of the present invention is particularly
suited for the detection of molecular interactions in a gas or
liquid phase environment at the membrane surface of the sensor. A
feature of the sensor is a drum-like cavity comprising a membrane
at the top which contacts the environment to be sensed, or more
walls that support the membrane, and a base with at least one
electrode. The harmonic response of the device is sensitive to the
surface properties of the membrane. The membrane also protects the
drive elements within the cavity from direct contact with the
environment. The cavity also has other elements and various sensor
embodiments will now be described in detail.
[0084] A resonant membrane sensor based on capacitive sensing is
shown in FIGS. 1a and 1b. Referring to the figures, the single
resonant membrane sensor 10 comprises a silicon wafer substrate 12,
a membrane 18, a circular planar electrode 14 located within the
substrate surface, and a spacer layer 16. The sensor cavity 22
comprises the resonating portion 28 of membrane 18, a circular
sidewall 20 that is formed as an opening in spacer layer 16, and a
base comprising the substrate 12 with planar electrode 14 formed
thereon.
[0085] The spacer layer 16 can be made of any electrically
insulating material with sufficient rigidity to maintain spacing
between the membrane and planar electrode during membrane movement.
The spacer layer can be prepared from silicon nitride, silicon
dioxide, and the like.
[0086] Circular planar electrode 14 is formed within wafer 12 by
diffusion or ion implantation. The sensor cavity 22 will generally
have an air dielectric, although other dielectrics may also be
utilized as application and design dictate. Lead 26 connects planar
electrode 14 to a voltage source (not shown).
[0087] Membrane 18 (and resonating portion 28) can be prepared from
electrically conductive material or non-conductive material. The
membrane 18 is a continuous sheet formed across the entire surface
of the sensor 10, the resonating portion 28 of membrane 18 is
circular in shape. This occurs because the membrane is supported by
a circular wall 20. The circular geometry of sensor membrane 18
distributes stress evenly and radially about the entire membrane
eliminating points of high intrinsic stress and can offer
preferably modes of oscillation as discussed herein. Choosing the
proper mode of excitation involves designating a mode spaced
sufficiently far from its neighbors such that cross mode
interference does not occur, that sufficient amplitude is obtained
and in which minimal damping occurs. Rectangular, square, or any
other geometry may be used as fabrication or application
dictates
[0088] Membrane 28 opposes planar electrode 14, form the opposing
conducting plates or electrodes of a capacitor, separated by the
cavity dialectic 22. Membrane 28 can be driven into resonance
electrostatically by charging the planar electrode 14 with a
variety of input functions such as sinusoids, square waves, saw
tooth waves, triangle waves, impulses, chirps, white noise, and the
like. A dc-bias voltage also may be simultaneously applied to tune
the mechanical and/or electrical response of the device. Membrane
18 and its resonating portion 28 can be grounded, preventing
unwanted electrochemical interaction between charges at the sensor
surface and the salts and biomolecules that may be present in test
solutions. In an alternative, membrane 18 does not have to be
grounded.
[0089] Decreasing nominal separation between planar electrode 14
and membrane 28 increases both the strength of electrostatic
actuation and the output signal (i.e., increased capacitance). This
increases sensitivity and decreases drive voltage requirements. In
a preferred embodiment, the separation is between about 0.25 to 2
microns. Contact of membrane 28 to planar electrode 14 results in
device failure, thus imposing a lower limit on separation.
[0090] The interior of cavity 22 is vented to outside atmosphere by
passageway or holes 24 traversing electrode 14 and substrate 12.
Venting eliminates pressure-related signal drift, such as long
timescale barometric effects, by equilibrating internal cavity
pressure with the outside atmosphere. Venting also minimizes short
timescale pressure gradients across the membrane due to acoustic
waves in the cavity. Vent surface area should be large enough to
allow adequate airflow into and out of the cavity during operation
yet must not compromise overall device performance (e.g., by
impacting the area of the planar electrode 14). Although four holes
are shown in FIG. 1, the number of holes and their diameter may
vary with the characteristics of the sensor and its intended
application. Venting may be eliminated entirely for some
applications.
[0091] Resonant micromechanical membrane sensor 50 in FIG. 2 is
similar overall to FIG. 1 but has a resonating membrane 56 that
does not extend to the sides of the sensor, the figure showing
minimal overlap with spacer layer 52 as compared to FIG. 1 where
membrane 18 extends fully over the spacer layer 16.
[0092] Microfabricated resonant membrane sensor 60 in FIG. 3, is
also similar to FIG. 1, but differs in having a layer 62 and having
side walls 66 formed of the same material as the membrane 64, due
to the conformal deposition of the membrane and the subsequent
removal of a sacrificial layer from underneath the membrane
resonating portion 68 compared to FIG. 1.
[0093] Capacitive detection of a resonating structure has
advantages over approaches using piezoelectricity or
piezoresistivity. For example, the simplest one-port device for
capacitive requires only a single electrode (see FIG. 1), while the
simplest piezoelectric and piezoresistive devices require a minimum
of two and three electrodes respectively and additional structures
such as piezoresistors and piezoelectric transducers. Capacitive is
more thermally stable than other transduction methods including
piezoresistivity and piezoelectricity. It is less affected by
temperature change than is piezoresistivity and piezoelectricity.
The temperature coefficients of resistivity of common
micromachining materials and pyroelectric constants of common
piezoelectric materials can be quite high. Capacitors, however,
exhibit extremely low temperature coefficients, are less noisy and
more sensitive than piezoelectric and piezoresistive devices
[0094] Detection may also be accomplished through alternative means
such as piezoelectricity, piezoresistivity, or optically when
capacitive means are not optimal. One example for optical detection
is provided in U.S. patent application Ser. No. 09/812,111, filed
Mar. 15, 2001, entitled "Method for Monitoring the Oscillatory
Characteristics of a Microfabricated Resonant Mass Sensor," and
incorporated herein by reference as if fully set forth herein.
[0095] The present resonant micromechanical membrane sensor can be
designed with a dedicated drive and sense electrode, separate from
the resonating membrane. In reference to FIG. 4, the lower portion
of the sensor 140 comprises substrate 142 with dual electrodes in a
concentric design. In a non-limiting example the outer electrode
144 can provide actuation and inner electrode 146 can provide
detection. Outer electrode 146 meets external voltage source at
contact 156 via lead 154. Inner electrode 146 goes to detection
circuitry via lead 148 and contact 150.
[0096] The concentric design of drive and sense electrodes in FIG.
4 provides optimal force and signal transduction. In operation, for
example, a drive signal, such as a harmonically varying sinusoid
with dc-offset, is applied through outer electrode 144. Magnitude
of induced charge acquired at inner electrode 146 is affected by
the displacement of the conductive resonating membrane of the
sensor. Electrode geometry can also be varied to excite other modes
of oscillation as desired. Separation between the concentric
electrodes must be sufficient (and/or appropriate shielding used)
to minimize stray fields and induced currents between the
electrodes.
[0097] The present micromechanical sensor can be designed with
electromechanical sense elements. In reference to FIG. 5, resonant
membrane force sensor 160, overall similar to the sensor in FIG. 1,
comprises a "circular-shaped" piezoresistive sense element 166 that
conforms to the outside border of circular-shaped resonating
membrane 164, where maximum stress occurs during membrane
oscillation.. The piezoresistive sense element 166 can be layered
above the membrane 164 or fabricated within the membrane as shown.
Sense element 166 can be prepared from doped silicon which has
piezoresistive qualities. In this embodiment, membrane 166 is
driven by electrostatic actuation (see discussion of FIG. 1) and
membrane displacement measured through the changes in the
resistance of the piezoresistive element 166. Change in resistance
can be determined by incorporating 166 via connections 168 and 170
into a Wheatstone bridge assembly.
[0098] In reference to FIGS. 6a and 6b, substrate 202 with lower
planar electrode 208 is the base of cavity 212 bounded on top by
electrode 210 directly affixed below resonating membrane 204 and
circular side wall 214. A thin ring of piezoelectric material 206,
such as PVDF, PZT, or ZnO, deposited locally above and around the
edges of the membrane 204 generates voltage when mechanically
stressed by movement of membrane 206 during electrostatic
actuation. By locating piezo-material 206 to the outside resonating
edge of membrane 204 where stress is greatest, sensitivity loss
from piezo-material mass and internal damping displacement is
reduced and signal acquisition maximized.
[0099] A metal upper counter electrode 210 together with the doped
lower electrode 208 . provides the charged plates for electrostatic
actuation. Membrane 206 can be surface micromachined from almost
any material, including polysilicon, silicon nitride, silicon
dioxide, and the like. The metal upper counter electrode 210 may be
deposited on a sacrificial layer prior to deposition of membrane
204. Piezoelectric voltage may be measured using amplification and
other techniques well known in the art. In the alternative a
piezoelectric ring can be used for actuation of the sensor with
capacitive detection.
[0100] Many approaches are possible for connecting the lower planar
electrode to a voltage source or ground. In FIG. 7, micromachined
resonant membrane sensor 280, planar electrode 284 connects at 288
to lead 286 which extend vertically through substrate 282 to emerge
at contact 290. Alternatively, in FIG. 8, micromachined resonant
membrane sensor 300, planar electrode 304 connects at 308 to lead
306 which extend horizontally through substrate 302 to emerge at
contact 310.
[0101] To further simply fabrication and operation, an alternative
embodiment of the device utilizes electrostatic drive transducers
and an external optical detection system. For example, the
detection circuitry can be eliminated from the sensor and replaced
with an optical sensor such as a laser Doppler vibrometer ("LDV").
LDV measures the oscillatory characteristics of the resonating
membrane by the effect of the membrane on the laser beam. U.S.
patent application Ser. No. 09/812,111, filed Mar. 15, 2001,
entitled "Method for Monitoring the Oscillatory Characteristics of
a Microfabricated Resonant Mass Sensor," and incorporated herein by
reference as if fully set forth herein, exemplifies the details of
using a Laser Doppler Vibrometer as a detection scheme in resonant
mass sensors. Other interferometers such as Michelson or
stroboscopic interferometers may also be used for this purpose.
[0102] Sensor Array Embodiments
[0103] The present invention includes a micromechanical resonant
membrane sensor array, which has various features of the individual
sensor embodiments described above. The sensors are microfabricated
to have nominally similar resonant frequencies and performance
characteristics except possibly in the case where the sensor unit
is used as a reference. Each sensor in the array needs to be spaced
an appropriate distance from its nearest neighbors or appropriately
isolated so that mechanical, acoustical, and electrical cross-talk
do not substantially propagate to the adjacent sensor sites.
[0104] In one embodiment, sensor array 400 shown in FIGS. 9a and 9b
comprises 12 separate sensor sites or units 414 similar in design
to the individual sensor unit shown in FIG. 1. Sensor array 400
comprises a silicon substrate 410 into which the individual planar
electrodes 416 are formed. Membrane 422 shown at the lower left in
FIG. 9a covers the entire substrate 412. The resonating membrane
424, above the sensor cavity (not shown), is part of membrane 422.
Spacer layer 425 shown at lower left is situated below membrane 422
and above substrate 412. The membrane 422 functions as both a
resonant element 424 and a barrier to isolate sample fluid from
contacting the drive elements 414. Membrane layer 422 is fabricated
of electrically conductive material and preferably as a single
continuous layer covering all the sensors in the array.
[0105] The resonating membrane for each sensor 424 is grounded by
membrane 422 contacting grounding strip 426 which has connecting
leads 428. Grounding of the exposed membrane surface 422 prevents
unwanted electrochemical interaction between charges at the sensor
surface and the salts and biomolecules in test solutions. A common
ground also reduces the number of discrete interconnects necessary
to address each sensor, which increases the number of channels
available for parallel actuation and detection the entire array. In
another embodiment discrete grounds may be used or the membranes
may not be grounded.
[0106] In sensor array 410, each sensor can be separately
interrogated by having a separate drive lead electrically isolated
from the other sensors. As seen in FIG. 9a, electrical isolation of
each sensor unit is accomplished 420, which represents a
non-conducting border material or a channel. FIGS. 9a and 9b
together show how the individual sensors can have separate drive
connections allowing individual sensor actuation and sensing. In
this regard, FIG. 9a shows planar electrode 416 having lead 418,
which extends downwards into the substrate, emerging on the
substrate 410 bottom side (418 in FIG. 9b). The unique position of
each contact point 418 allows for a separate connection to a
voltage source. Instead of individual sensor interrogation, one
skilled in the art would understand that groups of sensors can be
multiplexed such that a discrete number of individual sensors may
be simultaneously interrogated and the response simultaneously
measured.
[0107] Specific sensors sites in the array can be designed or
designated as a reference site. A reference site is a sensor in the
array that generates a control value to which sensors that measure
unknown are compared and extraneous variables, such as temperature,
fluctuations in pressure, environmental vibrations can be
eliminated. Sensitivity can be increased by using reference sensor
sites. Various types of reference sensors are contemplated. For
example, a reference sensor may be a sensor where the membrane is
fixed in position as a fixed plate capacitor such as when a
non-conductive dielectric support 430 is inserted into an otherwise
functioning sensor cavity. Support 430 may be prepared from silicon
dioxide, silicon nitride and the like. Other approaches also would
be apparent to one of ordinary skill in the art. In the case of
detecting chemical or biological compounds from a gas or liquid
environment, reference sites may also include be mechanically
active sensors that are chemically inactive, for example the sensor
does not bind a compound of interest. Other reference sensor sites
are possible and known to those of skill in the art.
[0108] Determining Membrane Dimensions
[0109] Membrane dimensions are dictated by a number of parameters,
primarily the desire to decrease damping, increase device
sensitivity, and the practical limits of fabrication.
[0110] Damping in acoustical MEMS-based sensors is present in four
major forms: internal material damping, assembly damping, viscous
damping and acoustic damping. The effects of damping are to
decrease device Q, decrease efficiency, and ultimately decrease
sensitivity. Among the four main contributors of damping, acoustic
damping is the dominant form of energy dissipation. Thus, membrane
size is driven primarily by the need to reduce acoustic
radiation.
[0111] When a resonating membrane sensor contacts a fluid
environment, the amount of acoustic propagation into the fluid and
the degree of acoustic coupling between the fluid and the membrane
relates to the acoustic wavelength of the surrounding medium at the
operating frequency and the membrane size. While acoustic
propagation can occur normal to the membrane surface, acoustic
damping may still be minimized by ensuring that membrane diameter
is always significantly smaller than the acoustic wavelength of the
immersion fluid at the operating frequency. Driving a membrane in
its fundamental mode will result in maximal signal amplitude and
minimal damping because the lower order modes have larger
displacements, lower resonant frequencies, and hence longer
acoustic wavelengths. Optimal membrane radii range from 2.5 to 50
microns. For these radii, the acoustic wavelength is smaller than
the membrane radius while operating in the fundamental mode.
[0112] Other modes of resonance may also be utilized. In some
cases, the higher order modes may increase device Q by offsetting
inertial effects and creating balanced modes of oscillation and/or
by reducing acoustic propagation by self-canceling of the acoustic
waves generated in the medium.
[0113] Internal material damping and assembly damping may be
minimized by proper material choice and device design. Single
crystal silicon is an excellent mechanical material due to its high
Young's modulus, low internal damping, zero residual stress, and
low coefficient of thermal expansion. This leads to devices with
high mechanical Q's and reliable operation. By eliminating features
such as contacting or friction surfaces, assembly damping may also
be minimized. Viscous damping is a small contributor of damping in
relatively inviscid fluids such as water.
[0114] Since membrane resonant frequency is highly dependant on the
membrane radius, membrane size is also limited by the desired
operating frequencies. The optimal operating frequencies from
mechanical and electrical standpoints lie in the kHz to low MHz
range. Above the low MHz range, signal processing components become
increasingly costly and complex and acoustic damping becomes a
major factor. At low frequencies, electrical 1/f noise dominates
and frequency shifts become difficult to detect.
[0115] Resonant membrane thickness is controlled by the desired
device sensitivity and fabrication limits. The membrane behaves in
a manner similar to a simple harmonic oscillator. Mass loading of
membrane surface increases the effective mass of the oscillator and
decreases the resonant frequency of the membrane. Device
sensitivity can be defined as the fractional change in resonant
frequency divided by the incremental increase in surface mass.
Algebraic rearrangement gives 1 S m = f F o m = - 1 2 M = - 1 2
t
[0116] where .DELTA.f is the mass-loaded resonant frequency shift,
F.sub.O is the unloaded resonant frequency, Am is the mass per unit
area of the added mass, M is the areal mass density, .rho. is the
membrane density, and t is the membrane thickness 25. Thinner
membranes give rise to increased sensitivity but practical
fabrication limitations sets membrane thickness to a minimum of
0.1-0.5 microns.
[0117] In the presence of fluid, an additional mass must be added
to compensate for mass loading due to the presence of fluid. This
mass of water can be approximated as a sphere with a radius equal
to that of the membrane. The sensitivity then becomes 2 S m = f F o
m = - 1 2 ( t + 4 3 r w )
[0118] where r is the radius, .rho..sub.w is the density of the
fluid and M=.rho.t. Sensitivity shows a direct correlation to
thickness. This is due to the inertia of the plate. In fluid,
thickness of the membrane has a decreased effect since the
mass-loading effect of the fluid dominates. FIGS. 10a and 10b
graphically illustrate the relationship between sensitivity and
thickness for a 10-micron radius Si membrane in air and water,
respectively. FIGS. 12a and 11b graphically illustrate resonant
frequency achieved in air and water, respectively, as a function of
membrane thickness and membrane radius. The results indicate that
small and thin membranes minimize damping and inertia. A membrane
thickness of about 0.1 to 0.5 microns and a membrane radius of
about 2.5 to 50 microns is preferred.
[0119] Fabrication of Preferred Embodiments
[0120] Various approaches may be used for to fabricate a resonant
micromachine membrane sensor of the invention. One approach is
shown in FIG. 12a -12f. A p-type, FZ silicon wafer with a
resistivity of >10,000 ohm cm is used as the substrate 82 and
electrode 84 and lead 86 are ion implanted at high energy and high
dose to a depth of 0.5 um and surface concentration of 1e16
ions/cm2. A 1 micron wet thermal [silicon dioxide] oxide layer 84
is grown and then patterned with a wet etch to define the spacer
layer 88 of about 1 micron in thickness 2000 A of silicon dioxide
is left un-etched as a subsequent etch stop. A backside align,
pattern, and DRIE (deep reactive ion etching) is used to form vent
holes 90 extending entirely through the material substrate 82 and
electrode 84. The sensor membrane is formed using a
silicon-on-insulator ("SOI") wafer 92, which comprises a silicon
"handle" layer 94, an intermediate layer of silicon dioxide or "box
oxide" 96 and a layer of silicon 98. The silicon layer 98 side of
SOI wafer 92 is fusion bonded to patterned spacer layer 88. The
handle wafer 94 and the "box" oxide layer 96 are removed by a wet
etch, leaving a membrane layer 100 (shown in exaggerated size).
Finally, vias are etched in the remaining oxide and metal is
patterned to form the final leads to the electrodes and contact
pads (not shown). Trenches or channels can also be etched to
physically and electrically separate the membrane or to separate
the sensors from each other in an array. Other types of silicon
wafers can be used in this process including a double polished
p-type silicon wafer.
[0121] In another approach, a 4'Si, DSP thin silicon wafer, either
p- or n-type is used as the substrate. 1 .mu.m of wet thermal oxide
is grown to passivate the wafer. 2000 A of a high temperature metal
such as titanium, tungsten or a titanium-tungsten composite is
deposited onto the surface and patterned to form electrodes and
leads. Vias are then etched into both the metal and oxide layers
straight down to the substrate. Next a 1.5 um layer of LTO or
phosphosilicate glass (PSG) is deposited by low pressure chemical
vapor deposition ("LPCVD"). A subsequent backside align, pattern,
and DRIE is used to form the vent holes. A CMP step is used to
planarize the LTO surface and reduce the surface roughness to a
magnitude favorable to bonding. After CMP, the oxide layer is
patterned with a wet etch to define the spacer layer and the
membrane formed from an SOI wafer as described in the first
approach.
[0122] Fabrication of a surface micromachined silicon nitride
membrane sensor is detailed as follows. Electrodes and lead lines
are diffused into a double-sided polished thin wafer using either
thermal diffusion or ion implantation. A thermal oxide layer is
then grown to act as an insulator and etch stop. A polysilicon
sacrificial layer is deposited using LPCVD or similar technique
such as PECVD. A second conformal tungsten electrode is then
deposited over the polysilicon layer and over vias to the lead
lines formed earlier. The membrane is formed by depositing low
temperature oxide (LTO) and/or low stress nitride (LSN) over the
tungsten electrode and polysilicon spacer layer. Vents are etched
from the backside of the wafer using DRIE. Finally, the sacrificial
polysilicon is removed, and the membrane released by a vapor phase
XeF.sub.2 etch process that isotropically removes silicon but is
extremely selective to silicon dioxide and certain metals, such as
for example aluminum.
[0123] The planar electrode may be fabricated by diffusion or ion
implantation of silicon doped with either boron or phosphorus. A
doped electrode of high impurity concentrations is useful to
minimize electrical resistance. In alternative embodiments,
electrodes may be patterned by metal deposition using techniques
such as evaporation, sputtering, or electroplating. Diffused
electrodes can withstand higher processing temperatures and harsher
processing conditions than their metal counterparts and are
preferred in many applications. However, they typically have
significantly higher electrical impedance's that can lead to
increased parasitic capacitance and signal degradation. In
applications where high sensitivity is required, metal electrodes
and leads may be preferred.
[0124] The above processes serve merely as examples of fabrication
that are among the many methods of microfabrication that can be
used to form devices of the invention. Further details can be found
among membrane-based pressure sensors and accelerometers. Due to
the parallel nature of microfabrication processes, these techniques
can be readily extended to microfabricate an array having many
sensors as described herein.
[0125] Modes of Operation of Preferred Embodiments
[0126] With reference to FIG. 13, in a preferred embodiment,
electrostatic actuation and capacitive detection are employed. Each
site in the sensor 111 is driven into resonance by white noise
source 110 applied through the lower electrode. As a result, the
membrane oscillates primarily in its fundamental mode. Applying a
constant voltage induces current in the electrode that is
proportional to the membrane impedance. At mechanical resonance,
the current component of the oscillation signal should be a
maximum. A band-pass filter 111 can be used to limit bandwidth.
Fast Fourier analysis 113 of the current signal produces peaks that
can be used to identify the resonant response of the system. A
constant current can also be applied to the membrane, and the
resultant voltages that are developed can be measured. Differential
measurements performed between derivatized sites and chemically
inactive sites can be used to compensate for temperature-induced
drift, non-specific adsorption, or noise due to external
vibrations, etc.
[0127] With reference to FIG. 14, in an alternative mode of
excitation and detection, the sensor 120 is incorporated as part of
a phase-locked loop (PLL) 126. A feedback-loop circuit 126
incorporating a phase comparator 123 sustains resonance of the
device 120 by locking on to the frequency at which a 90-degree
phase shift is maintained between the drive signal and the output
signal. The output signal passes through a low-pass filter 121 to
an amplifier 122 and then a phase comparator 123. The phase
comparator 123 adjusts the frequency of the voltage-controlled
oscillator 124 such that the frequencies of the input and output
signals match. Monitoring the frequency at which the PLL 126 is
locked with a frequency counter 125 provides a method a
continuously monitoring the resonant frequency.
[0128] In a yet alternative mode of excitation and detection,
harmonic sweeps of the excitation signal through frequencies
nominally bounding the resonant frequency are performed.
Ratiometric analysis of voltage division between a test site and a
fixed plate reference capacitor can be used to perform differential
measurements to decrease the effects of parasitic capacitances,
electronic noise, and drift. Phase information can also be utilized
to identify resonant peaks.
[0129] In a further embodiment, the prior device is integrated as
part of a tunable oscillator circuit. The electrical
characteristics of the circuit can be monitored to obtain
gain-phase information and device impedance data. Tunable
oscillator circuits provide a simple, inexpensive means of
maintaining resonant oscillations and when combined with further
circuitry such as sustaining amplifiers or automatic gain control
loops, can act as means for accurately exciting and monitoring
resonant elements.
[0130] The previous descriptions are meant to illustrate but not
limit the multiple modes of operation that can be utilized with a
single device. For example, similar variations of the above
schemes, with the proper adjustments, can be applied to alternative
devices with any combination electrostatic, piezoelectric, or
acoustic excitation and capacitive, piezoelectric, piezoresistive,
or optical detection.
[0131] Applications of Preferred Embodiments
[0132] The array of sensors is designed to operate with a parallel
array of molecular probes. Each site within the array can be
derivatized with a different molecular probe such that the device
becomes potentially chemically responsive to sample solutions. A
binding event between a substance in a sample solution and a
molecular probe results in an increase of the surface mass of the
membrane and a corresponding decrease in resonant frequency or
vibration. Screening is designed to be performed under wet
conditions and does not necessitate drying of the chip. Doing so
could alter the chemical reactivity of the involved species, cause
denaturing, conformational changes, or instabilities in the
substances, and create problems such as the precipitation of salt
from solution. For further application details, refer to U.S. Pat.
No. 5,912,181 entitled "Method for Molecular Detection Utilizing
Digital Micromirror Technology." Chemical binding constants and
affinity can be determined by titration of the sample solution over
the device and real-time monitoring of resonant frequency shifts as
a function of concentration. The chip is also robust enough to be
reusable such that multiple samples can be serially flowed over the
chip and screened in sequence.
[0133] With reference to FIG. 15, an application where such an
array would be useful is in pharmaceutical high throughput
screening (HTS). Activity of a molecule such as a receptor or
enzyme against an entire combinatorial library 130 can be performed
in parallel. Each member of the library 131 would be chemical bound
to an individual membrane 132. A solution containing the molecule
is passed over the entire chip. "Hits" are identified by locating
the sites that displayed mass-induced resonant frequency shifts.
Multiple screenings of various molecules against the same library
131 can be performed on a single derivatized chip by sequentially
flowing various test solutions containing the desired molecules and
wash solutions over the chip. Binding constants of hits can also be
measured by titration of samples.
[0134] Both individual sensors and sensor arrays can be used for a
variety of applications. This includes immobilizing a binding
partner such as a peptide, small molecule drug on the sensor and
testing for binding to a protein source such as human serum, or
immobilizing an array of binding partners and screening a phage
display library in solution. Another use is to immobilize nucleic
acid on the sensor membrane and then screen a solution analytes
that might be transcriptional factors such as activators or
repressors. Alternatively, transcription factors may be immobilized
to a sensor and evaluated for their ability to bind DNA or small
molecules. The sensors also can be used to identify and
characterize protein - protein interactions. This may include
specificity and affinity determination and involve screening
antibodies, drugs, determining binding between intracellular
mediators, lectin - lectin interactions, cell substrate
interactions, virus life cycle relevant interactions such as
integrase - nucleic acid binding, capsid protein - capsid protein
binding (i.e., viral assembly) and MRNA - protein binding (i.e.,
viral translational regulation). In another approach, small
molecule compounds such as drugs, mimetics, peptides can be
immobilized to the membrane and the sensor tested for binding to a
natural ligand.
[0135] While preferred embodiments and methods have been shown and
described, it will be apparent to one of ordinary skill in the art
that numerous alterations may be made without departing from the
spirit or scope of the invention. Therefore, the invention is not
limited except in accordance with the following claims.
* * * * *