U.S. patent application number 11/084306 was filed with the patent office on 2005-07-28 for method for detecting chemical interactions between naturally occurring biological analyte molecures.
This patent application is currently assigned to Affymetrix, INC.. Invention is credited to Forman, Jonathan E., Manalis, Scott R., Quate, Calvin F., Trulson, Mark O..
Application Number | 20050164289 11/084306 |
Document ID | / |
Family ID | 26727453 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050164289 |
Kind Code |
A1 |
Quate, Calvin F. ; et
al. |
July 28, 2005 |
Method for detecting chemical interactions between naturally
occurring biological analyte molecures
Abstract
A method of using micromechanical devices as sensors for
detecting chemical interactions between naturally occurring
bio-polymers which are non-identical binding partners is provided.
The method is useful whether the reactions occur through
electrostatic forces or other forces. Induced stress, heat, or
change in mass is detected where a binding partner is placed on a
cantilever for possible reaction with an analyte molecules (i.e., a
non-identical binding partner). The method is particularly useful
in determining DNA hybridization but may be useful in detecting
interaction in any chemical assay.
Inventors: |
Quate, Calvin F.; (Stanford,
CA) ; Trulson, Mark O.; (San Jose, CA) ;
Manalis, Scott R.; (Santa Barbara, CA) ; Forman,
Jonathan E.; (San Jose, CA) |
Correspondence
Address: |
AFFYMETRIX, INC
ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3380 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, INC.
Santa Clara
CA
|
Family ID: |
26727453 |
Appl. No.: |
11/084306 |
Filed: |
March 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11084306 |
Mar 18, 2005 |
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10072414 |
Feb 5, 2002 |
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10072414 |
Feb 5, 2002 |
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09097675 |
Jun 16, 1998 |
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6203983 |
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60049707 |
Jun 16, 1997 |
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Current U.S.
Class: |
435/6.12 ;
435/287.2 |
Current CPC
Class: |
G01N 33/54373 20130101;
C12Q 1/6816 20130101; Y10S 977/958 20130101; Y10S 977/80 20130101;
C12Q 1/6816 20130101; C12Q 2565/60 20130101; Y10S 977/924 20130101;
Y10S 977/957 20130101; Y10S 977/827 20130101; Y10S 977/835
20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
1-3. (canceled)
14. A method of detecting the interaction between naturally
occurring bio-polymers comprising: providing a gold support;
preparing a self assembled surface on the gold comprising
alkanethiol molecules; preparing the surface with a biopolymer;
introducing at least one bio-polymer analyte molecule to said
surface; and detecting a change based on binding between the
biopolymer and the biopolymer analyte.
15. A method as claimed in claim 14 wherein said bio-polymer
analyte molecules are DNA.
16. The method of claim 14 wherein said bio-polymer analyte
molecules are oligonucleotides, polynucleotides, or polyamino
acids.
17. The method of claim 14 wherein said interaction is a
non-covalent interaction.
18. A method of detecting the occurrence of a chemical interaction
between naturally occurring bio-polymers which are non-identical
binding partners comprising: providing a gold support; preparing a
self assembled surface on the gold comprising alkanethiol
molecules; preparing the surface with a first biopolymer material
which can act as a binding partner to a second bio-polymer
material; introducing said second biopolymer material; and
detecting a change based on binding between the first biopolymer
material and the second biopolymer material.
19. A method in accordance with claim 18 wherein the first
biopolymer material is a polynucleotide.
20. A method in accordance with claim 18 wherein the second
biopolymer material is a polynucleotide.
21. A method of detecting the chemical interaction between
naturally occurring bio-polymers comprising: providing a gold
support; preparing a self assembled polymeric surface on the gold;
preparing the surface with a biopolymer; introducing at least one
bio-polymer analyte molecule to said surface; and detecting a
change based on binding between the biopolymer and the biopolymer
analyte.
22. A method in accordance with claim 21 wherein the biopolymer
material is a polynucleotide.
23. A method of electronically sensing the interaction between
polynucleotide comprising: providing a gold support; preparing a
self assembled surface on the gold comprising alkanethiol
molecules; preparing the surface with a polynucleotide; contacting
at least one polynucleotide analyte molecule to said surface; and
detecting an electrochemical change based on binding between the
polynucleotide and the polynucleotide analyte.
24. The method of claim 23 wherein the sensing occurs without
modifying the analyte or the polynucleotide.
25. The method of claim 23 wherein the sensing is for biomedical or
environmental applications.
26. The method of claim 23 wherein there is a plurality of
polynucleotides to form an array.
27. A sensor apparatus for detecting binding between non identical
biopolymers; comprising: a gold support; a self assembled surface
on the gold comprising alkanethiol molecules; a biopolymer operably
connected to the self assembled surface; and a detector for
determining binding between the biopolymer and the biopolymer
analyte.
28. The sensor in accordance with claim 27, wherein the bioploymer
is an oligonucleotide, polynucleotide, or polyamino acid.
29. The sensor in accordance with claim 27, wherein the bioploymer
analyte is an oligonucleotide, polynucleotide, or polyamino
acid.
30. The sensor in accordance with claim 27, wherein the bioploymer
and the bioploymer analyte are oligonucleotides, polynucleotides,
or polyamino acids.
31. The sensor in accordance with claim 27, wherein the sensor is
arranged to produce an array.
Description
[0001] The present inventors claim priority to U.S. Provisional No.
60/049,707 filed Jun. 16, 1997, which is hereby incorporated by
reference for all it discloses and for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of
micromechanical devices as sensors for detecting physical or
chemical changes caused by chemical interactions between naturally
occurring bio-polymers which are non-identical binding partners,
such as can occur with polyamino acids, polynucleotides, and the
like. The method of the present invention is useful whether the
reactions occur through electrostatic forces or through other
forces. In particular, the present invention provides a method for
detecting chemical interactions between naturally occurring
bio-polymers which are non-identical binding partners where one
binding partner or probe molecule is placed on a cantilever for
possible reaction with a sample analyte molecule (i.e., a
non-identical binding partner). The physical or chemical change may
be induced stress, heat, or mass, for example. The present
invention is particularly useful in determining DNA hybridization
but may be used in detecting interactions between any analyte
molecules, whether monomeric or polymeric. Examples of polymer
arrays which can be used with the method of the present invention
include nucleic acid arrays, protein or polypeptide arrays,
carbohydrate arrays, and the like.
BACKGROUND OF THE INVENTION
[0003] As known in the art, various techniques have been used to
determine whether a chemical interaction has occurred between two
materials, such as between a probe carrying a binding partner and a
sample. In the specific example of determining whether DNA
hybridization has occurred, various techniques have been used to
extract information from a sample. For example, detection schemes
have been used that are responsive to fluorescence in order to
reveal specific interactions or hybridizations. U.S. Pat. No.
5,578,832, "Method and Apparatus for Imaging a Sample on a Device,"
issued to Trulson et al. ("the '832 patent") and U.S. Pat. No.
5,631,734, "Method and Apparatus for Detection of Fluorescently
Labeled Materials," issued to Stern et al. ("the '734 patent")
provide methods and systems for detecting a labeled marker on a
sample located on a support through the use of an excitation
radiation source and radiation optics. The '832 patent and the '734
patent are hereby incorporated by reference for all they disclose
and for all purposes. As described in the '832 and '734 patents,
these techniques employ the use of a label, for example, the DNA
probe is labeled with a fluorescent molecule, such as fluorophore
or biotin. Once the DNA probe is labeled according to prior
methods, an optical system can be used to determine whether
hybridization has occurred by measuring fluorescence activated
between the labeled sample and the probe material.
[0004] The present invention provides a method for determining
whether a chemical interaction has occurred between naturally
occurring bio-polymers which are non-identical binding partners
through detecting a physical or chemical change on a
micromechanical device called a cantilever. A cantilever, by way of
analogy, can be thought of as a diving board which has been reduced
to a very small size. More specifically, a cantilever is a physical
device that is attached to another object at one end and remains
free to move on the other end. Deflection or up and down movement
of the free end of the cantilever can then be detected. The method
of the present invention can be used with any chemical analyte to
generate a physical or chemical change, whether through affinity
binding, which may include hydrogen bonding, electrostatic
attractions, hydrophobic effects, dipole interactions, or through
other forces.
[0005] The use of micromechanical sensors is advantageous in the
method of the present invention for several reasons. Various
signals such as force, heat, stress, magnetism, charge, radiation
and chemical reactions can be readily transduced into a
micromechanical deflection by an appropriately coated structure,
such as a cantilever. In addition, silicon-based micromechanical
devices can easily be integrated into microelectronic processing
systems such as CMOS (Complementary Metal-Oxide-Semiconductor)- ,
as known to one of skill in the art. As a result, it is possible to
produce seamless sensors as low cost and to integrate them directly
into computers. Moreover, micromechanical sensors are very small,
typically approximately 400 .mu.m in length, approximately 40 .mu.m
wide and approximately 1 .mu.m thick. As a result, it is possible
to obtain a short response time, generally measured in
microseconds, as well as sensitivity superior to standard
techniques. Finally, it is possible to construct arrays of
micromechanical devices, thereby permitting complex analysis of a
variety of signals as well as the use of a variety of sensing
materials.
[0006] By way of background, it is known that stress induced by
self-assembled monolayers can be detected by observing the
deflection of a micromachined cantilever similar to those used in
the commercial Atomic Force Microscope ("AFM"), as described by
Berger et al., in "Surface Stress in the Self-Assembly of
Alkanethiols on Gold," Science, Jun. 27, 1997, Vol. 176, p. 2021
("Berger I"), which is hereby incorporated by reference for all it
teaches. The Berger et al. paper studied the surface stress changes
during self-assembly of selected molecules, including alkanethiol
molecules self-assembled on gold. The researchers found that the
stress increases linearly with the length of the alkyl chain of the
molecule. In addition, the researchers detected a change in the
state of stress with the formation of salt bridges formed when
mercaptohexadecanoic acid was deposited on a functionalized surface
coated with the self-assembled thiols. This change in cantilever
stress was used to detect the formation of the salt bridges when
the analyte molecules were introduced.
[0007] Other pertinent work involving michromechanical sensors is
reflected in a paper by Berger et al. entitled "Nanometers,
Picowatts, Femtojoules: Thermal Analysis and Optical Spectroscopy
Using Micromechanics," Analytical Methods & Instrumentation,
Special Issue, .mu.TAS '96 ("Berger II"), also incorporated by
reference for all it discloses and for all purposes. In Berger II,
examples of low-cost, disposable micromechanical devices are
described which perform optical absorption spectra, calorimetric
and thermal analysis, electrochemical stressograms, gas phase
adsorption and surface reaction monitors.
[0008] Other work in the area of micromechanical sensors is
reported by Ginzewski et al. in "Observation of a chemical reaction
using a micromechanical sensor," Chemical Physics Letters, Vol.
217, No. 5,6, Jan. 28, 1994, ("Gimzewski") which is hereby
incorporated by reference for all it discloses and for all
purposes. Gimzewski discloses a calorimeter for sensing chemical
reactions. The device is based on a micromechanical silicon lever
coated with a layer of aluminum. A sample is deposited on the lever
in a thin layer. Heat fluxes are detected by measuring the
deflection of the cantilever induced by the differential thermal
expansion of the lever. Specifically, Gimzewski discloses using
this technique to review the catalytic conversion of
H.sub.2+O.sub.2 to obtain H.sub.2O.
[0009] It is further known to operate multiple probes for the
atomic force microscope. As described by Minne et al., "Automated
parallel high-speed atomic force microscopy," Applied Physics
Letters, Volume 78, No. 18, May 4, 1998 ("Minne"), which is herein
incorporated by reference for all it discloses and for all
purposes, an expandable system is provided to operate multiple
probes for the atomic force microscope in parallel at high speeds.
The cantilever footprint is only 200 .mu.m wide which allows the
devices to be placed in a one-dimensional expandable parallel
array.
[0010] Yet another contribution to the art of micromechanical
sensors is described by Manalis et al., "Interdigital cantilevers
for atomic force microscopy," Applied Physics Letters, Vol. 69, No.
25, Dec. 16, 1996 ("Manalis I"), which is hereby incorporated by
reference for all it discloses and for all purposes. In Manalis I,
an AFM sensor is described in which a silicon cantilever is
micromachined into the shape of interdigitated fingers that form a
diffraction grating. When detecting a force, alternating fingers
are displaced while remaining fingers are held fixed. As a result,
a phase sensitive diffraction grating is created which allows the
cantilever displacement to be determined by measuring the intensity
of diffracted modes.
[0011] Another paper by Lang et al., "Sequential position readout
from arrays of micromechanical cantilever sensors," Applied Physics
Letters, Vol. 73, p. 383, 1998 ("Lang") describes using a reference
cantilever for canceling environmental noise. Lang is hereby
incorporated by reference for all it discloses and for all
purposes. In Lang, chemically specific responses are extracted in a
noisy environment using a sensor to detect specific chemical
interactions and an uncoated cantilever as a reference.
[0012] Finally, another paper by Manalis et al., "Two dimensional
micromechanical bimorph arrays for detection of thermal radiation,"
Applied Physics Letters, Jun. 16, 1997, (Manalis II) hereby
incorporated by reference for all it discloses and for all
purposes, describes fabricating arrays of cantilevers and using
them as sensitive detectors of head induced stress. Specifically,
the cantilevers described by Manalis II were placed on a grid with
50 microns on centers. The present inventors have determined that
this type of array is a suitable substrate for determining, for
example, hybridization.
SUMMARY OF THE INVENTION
[0013] Rather than using traditional labeling, such as optical or
electrochemical labeling, in order to detect chemical interactions
between naturally occurring bio-polymers which are non-identical
binding partners, the present inventors have determined a new and
useful method for "reading" a substrate to determine whether a
particular chemical interaction has occurred. In traditional
labeling, sample analyte molecules are modified in some way to
permit their detection when they combine with the probe molecules.
The method of the present invention is particularly useful in the
detection of hybridized sites on a DNA probe array. The method of
the present invention allows detection of hybridization without
modifying either the analyte or the probe molecules, i.e., it
requires no labeling.
[0014] According to the method of the present invention, a chemical
interaction between naturally occurring bio-polymers which are
non-identical binding partners is monitored by detecting a physical
or chemical change through deflection of a cantilever. The physical
or chemical change can be, for example, induced stress on the
cantilever which causes the cantilever to move or deflect. Standard
AFM techniques are then used to detect the deflection of the
cantilever. The physical or chemical change can also be in the form
of a heat reaction, which similarly causes the cantilever to
deflect or bend where the cantilever is made of two materials,
i.e., is a bimorph. A physical or chemical change might also result
in a change in mass on the cantilever. In such an example, the
resonant frequency of the cantilever will change due to the mass
change. Measuring the resonant frequency of the cantilever under
such circumstances will allow the physical or chemical change to be
detected.
[0015] In a specific embodiment of the present invention,
oligonucleotides are deposited onto a cantilever. The stress
induced by hybridization is detected with methods commonly used for
detecting cantilever deflection in the AFM. As is well known to one
skilled in the art, these methods are sensitive to the point where
deflections less than 0.01 nm can be easily detected. The substrate
used according to the method of the present invention allows
exploitation of the cantilever's properties in order to detect the
hybridized sites.
[0016] Specifically, the stress in the individual cantilevers is
monitored in the manner shown by Manalis II, noted above. First,
the surfaces of the cantilevers are prepared in the same manner now
common in immobilized sensor technology, as known to one skilled in
the art. Next, a binding partner, such as oligonucleotides, is
deposited on the cantilevers to form an array of probes. This
deposit will change the state of stress on the individual
cantilevers and this stress pattern is used as the reference. When
sample or analyte molecules (i.e., a non-identical binding partner)
are introduced to the cantilever and interact with the binding
partner (probe molecules) at appropriate sites, the stress on the
cantilever at the particular site will change as a result of the
interaction. The change in stress with the introduction of the
sample molecules will be monitored with standard AFM
techniques.
[0017] The present invention does not use optical or
electromechanical labels, as previously described. In addition, it
serves as a tool for understanding the processes involved in
chemical interactions between naturally occurring bio-polymers
which are non-identical binding partners, such as DNA
hybridization, by providing additional ways to measure events such
as the length of the chemical interaction and the number of
molecules hybridized. Moreover, it provides an additional, highly
sensitive, low-cost means to monitor chemical interactions, as
described in detail below.
DETAILED DESCRIPTION OF THE INVENTION
[0018] According to the present invention, a physical or chemical
change on a cantilever is measured in order to monitor the
occurrence of a chemical interaction between naturally occurring
bio-polymers which are non-identical binding partners, for example,
between biological polymers or other analytes, monomeric or
polymeric. Typically, the cantilever as used with the method of the
present invention is approximately 100 .mu.m in length, 50 .mu.m in
width and approximately 1 .mu.m in thickness. When a chemical
interaction occurs on the cantilever, a physical or chemical change
occurs causing the cantilever to be deflected, i.e., moved up or
down at its free end. Such deflection motion can be detected to a
very fine degree, for example, up to a fraction of a diameter of an
atom.
[0019] Turning to the specific example of using the present method
to detect DNA hybridization, as shown in FIG. 1, the surface of a
cantilever 110 is first prepared in order to be able to attach
single strands of DNA. Such surface preparations are known to those
of skill in the art of DNA hybridization detection methods. More
specifically, cantilevers made of a solid substrate, for example
silicon or similar materials, are prepared with special surfaces of
silicon dioxide (SiO.sub.2) and standard procedures are used for
making a functionalized layer that allows attachment of probe
molecules. Next, a binding partner or probes molecules, for
example, single stranded DNA 120, are introduced onto one surface
of the cantilever.
[0020] As shown in FIG. 2, the device is then preferably mounted
into a liquid cell 130, for example, containing an aqueous buffer
140. A detector 150 is employed in which a laser beam 160 is shown
on the cantilever and reflects off of the cantilever. The reflected
spot 170 of light is used to determine the relative position of the
cantilever. In other words, movement of the cantilever can be
determined by directly detecting the movement or angle of the
reflected laser beam light. This provides a particular advantage in
the present method in that it is always possible to obtain a strong
signal from the reflected light.
[0021] The response from this first deflection on the detector is
used as a reference to determine cantilever deflection, as further
described. Next, sample analyte molecules, such as DNA is
introduced to the surface of the cantilever containing single
stranded DNA. The sample analyte molecules will hybridize with
selected strands of DNA on the cantilever, as reflected at numeral
180 in FIG. 3. As a result, stress is induced on the cantilever
which will cause the cantilever to deflect. More specifically, when
hybridization occurs, surface pressure results by the addition of
negative charges on the surface of the cantilever because DNA is a
polyanion. In other words, hybridization causes more electrostatic
charges to build up on the cantilever surface which tend to repel
one another. Because the sample analyte molecules are only on one
surface of the cantilever, the surface of the cantilever deflects
due to this repelling action. This deflection will appear on the
deflector as a signal 170' in FIG. 3 which can be correlated
against the reference signal. It should be noted that the method of
the present invention can be used with negatively charged analytes
(such as DNA) or positively charged analytes. In addition, the
method of the present invention can also be used with uncharged
analytes because forces other than electrostatic forces, such as
dipole forces, can be employed with the present method.
[0022] The detector used with the present invention can be any
optical detector capable of tracking reflected laser light as known
to one of ordinary skill in the art, for example, can be a split
photodiode, linear array of photodetectors, piezo resistance
elements or the like.
[0023] In an alternative embodiment, shown in FIG. 4, a second
cantilever 190 can be used as a reference cantilever. The second
cantilever 190 is preferably mounted side by side with cantilever
110. In such an embodiment, a surface of the second cantilever 190
is prepared in the same manner as the first cantilever 110 which
will be used for hybridization. However, the second cantilever 190
does not have a binding partner, such as single stranded DNA,
attached onto one of its surfaces and is not treated with sample
analyte molecules, such as DNA. In this case, one signal (numerals
170 and 170' in FIG. 4) from each cantilever is detected by the
detector and the difference between the reflected light between the
two cantilevers is analyzed. The embodiment shown in FIG. 4 cancels
any spurious motion of the cantilever caused, for example, by the
environment, such as the liquid in the container.
[0024] In either embodiment, the signals detected by the detector
are then analyzed in order to determine whether hybridization, for
example, has occurred. If there is a change in position after the
sample analyte molecules, such as DNA, have been introduced on the
single cantilever (single cantilever embodiment), or if the
cantilever carrying the sample analyte molecules, such as DNA, has
changed its position in relation to the reference cantilever (two
cantilever embodiment), hybridization has been detected.
[0025] In yet another embodiment, several pairs of cantilevers
could be used, with one cantilever carrying a specific probe
molecules and the other cantilever of the pair carrying a
non-specific probe molecule or no probe molecule at all. In the
context of DNA hybridization, for example, several pairs of
cantilevers could be used each carrying a different sequence of
single stranded DNA. Multiple pairs of cantilevers organized in
such a fashion are known as an array of cantilevers. In an array,
each cantilever pair includes one cantilever for hybridization and
one neutral or reference cantilever. The difference between signals
of each cantilever pair in the array provides the true
hybridization signal for that pair, similar to the system described
with respect to FIG. 4.
[0026] With a cantilever array, it is possible to introduce a
complex mixture of molecules into the liquid flow cell encompassing
the array and to identify those molecules in the complex by
determining which cantilevers hybridize. The number of cantilever
pairs which can be used in an array is unlimited. Such a cantilever
array has practical utility in both biomedical and environmental
applications. An example of an environmental application would be
to use such a detector to identify an unknown contaminant in a
sample of air or water which might have been infected by
environmental terrorists. The possible applications for the method
of the present invention are limitless.
[0027] In still another alternative embodiment, an interdigital
array of cantilevers, as described above by Manalis I, can be used
in the method of the present invention. In an interdigital
cantilever array, interleaved fingers are built onto a cantilever
in the form of a grid. The cantilever deflects one pair of fingers
while the other remains stationery.
[0028] The method of the present invention is not limited to the
particular embodiments disclosed herein and can be employed to
detect any chemical interaction between naturally occurring
bio-polymers which are non-identical binding partners with accuracy
and at a low cost.
* * * * *