U.S. patent application number 10/842512 was filed with the patent office on 2004-10-21 for methods and compositions for directed microwave chemistry.
Invention is credited to Martin, Mark T..
Application Number | 20040209303 10/842512 |
Document ID | / |
Family ID | 46301291 |
Filed Date | 2004-10-21 |
United States Patent
Application |
20040209303 |
Kind Code |
A1 |
Martin, Mark T. |
October 21, 2004 |
Methods and compositions for directed microwave chemistry
Abstract
The present invention concerns a novel means by which chemical
preparations can be made. Reactions can be accelerated on special
cartridges using microwave energy. The chips contain materials that
efficiently absorb microwave energy causing chemical reaction rate
increases. The invention is important in many chemical
transformations including those used in protein chemistry, in
nucleic acid chemistry, in analytical chemistry, and in the
polymerase chain reaction.
Inventors: |
Martin, Mark T.; (Rockville,
MD) |
Correspondence
Address: |
LINIAK, BERENATO & WHITE, LLC
6550 ROCK SPRING DRIVE
SUITE 240
BETHESDA
MD
20817
US
|
Family ID: |
46301291 |
Appl. No.: |
10/842512 |
Filed: |
May 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10842512 |
May 11, 2004 |
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10234092 |
Sep 5, 2002 |
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10234092 |
Sep 5, 2002 |
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09968517 |
Oct 2, 2001 |
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60237192 |
Oct 3, 2000 |
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Current U.S.
Class: |
435/6.11 ;
435/7.1 |
Current CPC
Class: |
C12N 11/00 20130101;
G01N 33/54393 20130101; C12N 13/00 20130101; G01N 33/5436
20130101 |
Class at
Publication: |
435/006 ;
435/007.1 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/542 |
Claims
What is claimed is:
1. A method for accelerating a chemical reaction involving one or
more reactant(s) and a carrier fluid, said method comprising: (a)
providing a composite material to said reactant(s) and carrier
fluid, said composite material comprising a solid material
susceptible to dielectric heating, (b) applying an electromagnetic
field to said composite material, said electromagnetic field being
sufficient to result in dielectric heating of said solid material,
and (c) allowing said composite material to transfer heat to said
reactant(s); (d) whereby product is formed from said reactant(s),
thereby accelerating said chemical reaction.
2. The method of claim 1, wherein said chemical reaction comprises
a biospecific binding reaction.
3. The method of claim 1, wherein one or more said reactant(s) is a
protein molecule.
4. The method of claim 1, wherein one of said one or more
reactant(s) is an antibody or a fragment of an antibody that
retains the biospecific binding specificity of the antibody.
5. The method of claim 1, wherein the said chemical reaction is
between an antibody and its antigen.
6. The method of claim 1, wherein one or more said reactant(s) is a
nucleic acid molecule.
7. The method of claim 1, wherein said reaction comprises annealing
two nucleic acid molecules to one another.
8. The method of claim 1, wherein the chemical reaction comprises a
nucleic acid amplification reaction.
9. The method of claim 8, wherein the amplification reaction is a
polymerase chain reaction.
10. The method of claim 1, further comprising the step of measuring
the extent or rate of said chemical reaction.
11. The method of claim 1, further comprising the steps of: (e)
contacting said composite with one or more reactant(s) capable of
participating in a biospecific interaction with a reactant (a), or
a product produced as a consequence of a chemical reaction
involving a reactant (a), (f) allowing said additional reactant(s)
to react in said biospecific interaction, and (g) measuring the
extent or rate of said biospecific interaction.
12. The method of claim 1, further comprising the acceleration of
one or more additional reactions, said method comprising the
additional steps of: (e) contacting said composite with one or more
additional reactant(s), wherein said reactant(s) are capable of
participating in one or more further chemical reactions involving a
reactant (a), or a product produced as a consequence of a chemical
reaction involving a reactant (a), (f) applying an electromagnetic
field to said composite, said electromagnetic field being
sufficient to result in dielectric heating of said solid material,
said additional reactant(s) being heated by heat transfer from said
heated solid material, and (g) allowing said heated additional
reactant(s) to react with either a reactant (a), or a product
produced as a consequence of a chemical reaction involving a
reactant (a), thereby accelerating said one or more additional
chemical reactions.
13. The method of claim 12, further comprising the step of
measuring the extent or rate of said chemical reaction.
14. The method of claim 12, wherein said one or more additional
reaction(s) comprises the annealing of two nucleic acid
molecules.
15. The method of claim 12, wherein said one or more additional
reaction(s) comprises a nucleic acid amplification reaction.
16. The method of claim 15, wherein said amplification reaction is
a polymerase chain reaction (PCR).
17. The method of claim 1, wherein the wavelength of the applied
field is between 1 cm and 100 m.
18. The method of claim 12, wherein the wavelength of the applied
field is between 1 cm and 100 m.
19. A composite comprising a solid material responsive to
dielectric heating having a surface molecule capable of biospecific
interaction with a reactant molecule.
20. The composite of claim 19, wherein said surface molecule is
bound or immobilized to said surface via a noncovalent adsorption
reaction.
21. The composite of claim 19, wherein said surface is capable of a
covalent conjugation reaction with said reactant molecule.
22. The composite of claim 19, wherein said surface is selected
from the group consisting of a microarray chip, a macroarray chip,
a test tube, a Petri dish, and a microtiter plate.
23. An instrument that emits electromagnetic radiation sufficient
to accelerate a chemical reaction, wherein said chemical reaction
involves (a) providing a composite material to said reactant(s) and
carrier fluid, said composite material comprising a solid material
susceptible to dielectric heating, (b) applying an electromagnetic
field to said composite material, said electromagnetic field being
sufficient to result in dielectric heating of said solid material,
and (c) allowing said composite material to transfer heat to said
reactant(s); (d) whereby product is formed from said reactant(s),
thereby accelerating said chemical reaction.
24. The instrument of claim 23, wherein said chemical reaction
comprises the annealing of two nucleic acid molecules.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/234,092 (filed on Sep. 5, 2002), which
application is a continuation in part of U.S. patent application
Ser. No. 09/968,517 (filed on Oct. 2, 2001), which application
claims priority to U.S. patent application Ser. No. 60/237192
(filed on Oct. 3, 2000, now abandoned), all of which applications
are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of microwave
chemistry. It also relates to the field of biotechnology,
specifically microplate- and array chip-based preparative and
analytical chemistry.
BACKGROUND OF THE INVENTION
[0003] Until now, no one has performed chemical transformations as
disclosed herein. Devices are used that emit
radiofrequency/microwave energy. The energy is directed to a target
object, for example, a microarray chip or a microtiter plate that
contains one or more material(s) that absorb(s) microwave energy.
The microwave-generated heat energy accelerates a desired chemical
reaction on or near the surface of the targeted object.
[0004] Microwave Chemistry
[0005] Microwaves (including radiofrequency or RF electromagnetic
radiation) are commonly used in wireless communication devices.
Advances in microwave transmission have improved along with
tremendous recent technological improvements in the satellite and
communications industry (for example, in cell phones and wireless
internet).
[0006] Microwaves are also well known in common kitchen appliances.
Microwave ovens heat water-containing food rapidly because water is
efficient at converting microwave energy to thermal energy. Kitchen
microwave ovens emit microwaves at a frequency of 2.45 GHz, which
is well within the microwave absorption spectrum of water.
Frequencies outside of the absorption spectrum of water would not
heat food as well.
[0007] Another use for microwave heating is in chemical reaction
applications (Bose et al., 1997; Bradley, 2001; Wathey et al.,
2002; Lew et al., 2002). Microwave chemistry refers to the use of
microwaves to accelerate chemical reactions. Reactions are usually
carried out using microwave radiation to heat bulk solutions that
contain the reactants (Mingos & Baghurst, 1991; Zlotorzynski,
1995). Often these reactions are performed in non-aqueous solvents.
Microwave ovens specifically designed for use in carrying out
microwave chemistry of bulk reaction solutions are commercially
available (CEM Corporation (Mathews, N.C.), Milestone, Inc.
(Monroe, Conn.), Personal Chemistry AB (Uppsala, Sweden),
PerkinElmer Instruments (Shelton, Conn.)).
[0008] Microwave accelerated reactions are sometimes run on
solvent-free supports such as alumina and silica (Varma, 2001;
Bose, 1997; Bram et al., 1990). The supports can be doped with
reagents, for example in detoxifying waste. The supports are chosen
because they are inexpensive and recyclable agents which
non-specifically adsorb/extract the reagent of interest. No
specific binding, such as by antibodies or nucleic acids, is used
to capture reagents.
[0009] Another example of the application of microwaves to
accelerate chemical reactions is the use of microwave-absorbing
particles to enhance the heating of a bulk solution (Holworth et
al., 1998). In this case, dispersed cobalt and magnetite
nanoparticles were used as microwave (2.45 GHz) absorbers to heat a
bulk xylene solution. Xylene is a non-polar solvent not appreciably
heated by microwaves at 2.45 GHz. In one such case, microwaves were
used to accelerate the rate of an enzyme-catalyzed reaction (Kidwai
et al., 1998). In another case, Milestone, Inc. (Monroe, Conn.)
sells microwave-absorbing/heating composites of PTFE and graphite
which are designed to be dropped into test tubes to accelerate
microwave heating of solutions during chemical syntheses. However,
in these cases the microwaves are not directed to heat a surface,
but used to heat the bulk solution.
[0010] In another application, microwaves have been used to heat
the bulk solvent during solid-phase combinatorial chemistry (Kappe,
2001; Bradley, 2001; Lidstrom et al., 2001; Blackwell, 2003). In
these cases, conventional resins (polystyrene, for example)
function as solid scaffolds for chemistry. The bulk solution was
the target of the microwave heating.
[0011] In another case, microwaves were used to accelerate a
chromogenic reaction between noble metals and chromogenic reagents.
This analytical reaction was performed in solution by flow
injection analysis (FIA) (Jin et al., 1999). The reaction depended
on bulk solvent heating rather than targeted dielectric material
heating.
[0012] In yet another case, microwaves were used to enhance the
solution phase formation of a fluorescent complex of aluminum
(Kubrakova, 2000). The fluorescence intensity could be used to
measure aluminum ions in solution. Again, the reaction depended on
bulk heating of solvent.
[0013] In yet other cases, microwave heating has been used in
biochemistry applications. Microwave heating has been used to
assisted in protein staining (Nesatyy et al., 2002; Jain, 2002).
Bulk microwave heating of samples has been used to accelerate
antibody-antigen binding reactions in immunoassays,
immunohistochemical assays, and DNA in-situ hybridization assays
(Leong & Milios, 1986; Hjerpe et al., 1988; van den Kant et
al., 1988; Boon & Kok, 1989; Kok & Boon, 1990; van den
Brink et al., 1990; Slap 2003). In another example, microwaves were
used to accelerate the enzymatic synthesis of oligosaccharides
(Maugard et al., 2003). In another instance, microwaves were used
as a heat source during PCR (Fermer et al., 2003). In none of these
instances was microwave heating directed to a solid surface, but
rather microwave heating was applied to heat a bulk aqueous
target.
[0014] The present invention discloses a novel means of using
microwave energy to accelerate specific chemical reactions on or
near a microwave susceptible material. Reaction specificity comes
from the fact that the reactants (or molecules that form the
reactants) are biomolecules capable of biospecific interactions.
Microwave irradiation causes a temperature increase in the
microwave susceptible material, which consequently causes a
reaction rate increase of the reactants to form products.
OBJECTS OF THE INVENTION
[0015] The invention is directed toward an improved process and
apparatus for accelerating the rate of specific chemical reactions.
It is another objective of the invention that the accelerated
reactions be highly controllable, so that they can be selectively
turned on or off, or be modulated, by a user at will. It is yet
another objective of this invention to efficiently direct microwave
heating to the surfaces of bioanalytical array chips and microtiter
plates to accelerate preparative and analytical reactions. A
further objective of the invention is to provide such improved
reaction rates and specificity to a diverse number and type of
analytical and preparative chemical reactions. It is yet another
objective of this invention to accelerate biospecific binding
interactions, such antibody-antigen binding and hybridization of
nucleic acids. It is yet another objective of this invention to
perform polymerase chain reaction (PCR) amplification of nucleic
acids.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIGS. 1a-1c: Planar surfaces for directed microwave
chemistry (e.g., on a microarray chip or in the well of a
microtiter plate). The surface on which the reaction occurs may
contain the dielectric as shown in FIG. 1a or be adjacent to the
dielectric, as shown in FIG. 1b and FIG. 1c. The dielectric can be
a part of a disposable reaction cartridge (e.g., part of an array
chip or microtiter plate) as in FIGS. 1a and 1b, or the dielectric
may be a permanent part of the microwave-generating instrument as
in FIG. 1c.
[0017] FIG. 2: A Microwave Accelerated Targeted Triggered Reaction
("MATTR") instrument. The components of the instrument are a
microwave generator (such as a magnetron), a reaction cavity (e.g.,
oven or waveguide) with a built-in holder for MATTR disposable
chips. The instrument may also include features such as temperature
monitor (thermocouple or IR thermometer) and a light sensor such as
a PMT or a CCD camera. Microwave generation (power, time, pulsing,
etc.) can be controlled by computer. The computer can also be used
to control/monitor temperature and record and analyze light
acquisition.
[0018] FIG. 3: Structures of MATTR Chemiluminescent Compounds.
[0019] FIG. 4: Microwave components of a waveguide-based MATTR
instrument. The figure includes the microwave generator and
waveguide.
[0020] FIG. 5: Microwave components of the MATTR instrument shown
in FIG. 4. The figure includes a close-up of the waveguide.
[0021] FIG. 6: MATTR-based sandwich immunoassays for TNF.alpha..
Upon microwave irradiation, light is emitted from; (A) CL labels
indirectly bound to the analyte, TNF.alpha., or (B) from
enzyme-generated CL labels.
[0022] FIG. 7: Microwave-accelerated protein adsorption to
microwell plates.
[0023] FIG. 8: Microwave-accelerated specific antibody-antigen
binding (microwave-induced chemiluminescence detection).
[0024] FIG. 9: Microwave-accelerated specific antibody-antigen
binding (fluorescence detection).
[0025] FIG. 10: A typical nucleic acid assay.
[0026] FIG. 11: Microwave-accelerated specific nucleic acid
hybridization used in an assay for Human Actin mRNA: 1. Actin mRNA,
10 min, 65.degree. C. water bath; 2. Actin mRNA, 60 min, 65.degree.
C. water bath; 3. Actin mRNA, 10 min, microwave; 4. Control Cox-2
mRNA, 60 min, 65.degree. C. water bath; 5. Control Cox-2 mRNA, 10
min, microwave; 6. Control no mRNA, 60 min, 65.degree. C. water
bath; 7. Control no mRNA, 10 min, microwave.
[0027] FIG. 12: Polymerase Chain Reaction (PCR).
SUMMARY OF THE INVENTION
[0028] The invention describes a means in which chemical reactions
(catalytic or stoichiometric) can be accelerated by targeted
microwaves. The invention has been given the acronym "MATTR", which
stands for Microwave Accelerated Targeted Triggered Reactions. The
reactions preferably occur on or near solid surfaces (hereinafter
collectively referred to as "solid supports"). Suitable solid
supports contain a microwave absorbing material, which heats upon
absorbing microwaves. Reactants may be covalently or non-covalently
attached to the surface, or they may be within thermal proximity of
the microwave absorber, but not attached to the surface. For
example, the reactants may be on the surface of a microchip or in
the well of microtiter plates used for bioanalytical reactions. The
microwave instrument power, frequency, and duration of the
microwave emission are pre-determined in the laboratory. Following
microwave heating, a change in the reagent may be noted by a
physico-chemical change that takes place in the formation of
product(s) from reactant(s). The specific chemical rate
acceleration can be used for preparative and/or analytical
applications. In analytical applications, the reaction may
optionally be monitored and/or quantitated, for example in medical
diagnostics, by an accompanied observable physico-chemical change
(color change, for example). In preparative applications, the
presence of a microwave-dielectric layer can assist in surface
chemistry to prepare the solid support for subsequent analytical
reactions or be used to accelerate heat-dependent molecular binding
and amplification reactions.
[0029] In detail, the invention provides a method for accelerating
a chemical reaction involving one or more reactant(s) and a carrier
fluid, the method comprising:
[0030] (a) providing a composite material to the reactant(s) and
carrier fluid, the composite material comprising a solid material
susceptible to dielectric heating,
[0031] (b) applying an electromagnetic field to the composite
material, the electromagnetic field being sufficient to result in
dielectric heating of the solid material, and
[0032] (c) allowing the composite material to transfer heat to the
reactant(s);
[0033] (d) whereby product is formed from the reactant(s), thereby
accelerating the chemical reaction.
[0034] The invention further concerns the embodiments of such
method wherein the chemical reaction comprises a biospecific
binding reaction.
[0035] The invention further concerns the embodiments of such
methods wherein one or more the reactant(s) is a protein molecule,
or an antibody or a fragment of an antibody that retains the
biospecific binding specificity of the antibody or a nucleic acid
molecule.
[0036] The invention further concerns the embodiments of such
methods wherein the chemical reaction is between an antibody and
its antigen.
[0037] The invention further concerns the embodiments of such
methods wherein the reaction comprises annealing two nucleic acid
molecules to one another. The invention further concerns the
embodiments of such methods wherein the chemical reaction comprises
a nucleic acid amplification reaction (especially a polymerase
chain reaction).
[0038] The invention further concerns the embodiments of such
methods further comprising the step of measuring the extent or rate
of the chemical reaction.
[0039] The invention further concerns the embodiments of such
methods further comprising the steps of:
[0040] (e) contacting the composite with one or more reactant(s)
capable of participating in a biospecific interaction with a
reactant (a), or a product produced as a consequence of a chemical
reaction involving a reactant (a),
[0041] (f) allowing the additional reactant(s) to react in the
biospecific interaction, and
[0042] (g) measuring the extent or rate of the biospecific
interaction.
[0043] The invention further concerns the embodiments of such
methods further comprising the acceleration of one or more
additional reactions, the method comprising the additional steps
of:
[0044] (e) contacting the composite with one or more additional
reactant(s), wherein the reactant(s) are capable of participating
in one or more further chemical reactions involving a reactant (a),
or a product produced as a consequence of a chemical reaction
involving a reactant (a),
[0045] (f) applying an electromagnetic field to the composite, the
electromagnetic field being sufficient to result in dielectric
heating of the solid material, the additional reactant(s) being
heated by heat transfer from the heated solid material, and
[0046] (g) allowing the heated additional reactant(s) to react with
either a reactant (a), or a product produced as a consequence of a
chemical reaction involving a reactant (a), thereby accelerating
the one or more additional chemical reactions.
[0047] The invention further concerns the embodiments of such
methods further comprising the step of measuring the extent or rate
of the chemical reaction.
[0048] The invention further concerns the embodiments of such
methods wherein the one or more additional reaction(s) comprises
the annealing of two nucleic acid molecules. The invention further
concerns the embodiments of such methods wherein such one or more
additional reaction(s) comprises a nucleic acid amplification
reaction (especially a polymerase chain reaction (PCR).
[0049] The invention further concerns the embodiments of such
methods wherein the wavelength of the applied field is between 1 cm
and 100 m.
[0050] The invention further concerns a composite comprising a
solid material responsive to dielectric heating having a surface
molecule capable of biospecific interaction with a reactant
molecule.
[0051] The invention further concerns the embodiments of such
composite wherein the surface molecule is bound or immobilized to
the surface via a noncovalent adsorption reaction.
[0052] The invention further concerns the embodiments of such
composite wherein the surface is capable of a covalent conjugation
reaction with the reactant molecule.
[0053] The invention further concerns the embodiments of such
composite wherein the surface is selected from the group consisting
of a microarray chip, a macroarray chip, a test tube, a Petri dish,
and a microtiter plate.
[0054] The invention further concerns an instrument that emits
electromagnetic radiation sufficient to accelerate a chemical
reaction, wherein the chemical reaction involves
[0055] (a) providing a composite material to the reactant(s) and
carrier fluid, the composite material comprising a solid material
susceptible to dielectric heating,
[0056] (b) applying an electromagnetic field to the composite
material, the electromagnetic field being sufficient to result in
dielectric heating of the solid material, and
[0057] (c) allowing the composite material to transfer heat to the
reactant(s);
[0058] (d) whereby product is formed from the reactant(s), thereby
accelerating the chemical reaction.
[0059] The invention further concerns the embodiments of such
instrument wherein the chemical reaction comprises the annealing of
two nucleic acid molecules.
[0060] Definitions
[0061] Accelerate: To increase the rate of a chemical reaction,
preferably by at least 10%, more preferably by at least 50%, and
most preferably by at least 100% or more.
[0062] Aqueous Solution: A liquid medium that is more than 50%
water by volume.
[0063] Biospecific Binding Reaction (Biospecific Interaction): The
contact of a biological molecule to a biological or non-biological
molecule via three or more spatially distinct physical
interactions. The interactions are typically van der Waals
interactions, hydrogen bonds, and ionic interactions. Biospecific
interactions may also involve covalent bonds.
[0064] Cartridge: A vessel or device in which a reaction takes
place. The cartridge may be coated with a dielectric. Examples of
cartridges are microarray chips and microtiter plates.
[0065] Chemical Reaction: The chemical transformation of one or
more molecules (reactant(s)) to form one or more molecules
(product(s)). The definition includes covalent (such as hydrolysis)
and noncovalent (such as binding events) transformations.
[0066] Chip: An essentially planar object that has one or more
zones on its surface for desired chemical reactions to take place.
A chip is preferably small enough and light enough to be held in
one hand. If biological molecules are involved in the reactions,
the chip is also known as a biochip.
[0067] Composite: A solid made of two or more distinct types of
materials or molecules. If a composite is made of multiple
materials, the materials may be blended or physically distinct. If
physically distinct, the materials may be irreversibly joined
(e.g., glued together) or reversibly joined (e.g., snapped
together).
[0068] DNA: Deoxyribonucleic acid, usually 2'-deoxy-5'-ribonucleic
acid. The sequence of nucleotide residues of DNA can comprise genes
that can encode proteins. Cells possess the capability to read this
code to form proteins.
[0069] Dielectric Heating: Heating of a dielectric
(electrically-insulatin- g) material by electromagnetic radiation
in the wavelengths between approximately 5 cm and 100 m.
[0070] Hybridization: Coming together (annealing) of
single-stranded nucleic acid sequences by hydrogen bonding of
complementary bases to form double-stranded molecules; this process
is the basis for molecular biological techniques in which a labeled
probe oligonucleotide is used to detect a polynucleotide or
oligonucleotide possessing the identical or similar sequence (e.g.,
Southern hybridization, Northern hybridization). Hybridization is
also critical in PCR amplification of nucleic acids.
[0071] Lossy Material: A (dielectric) material that loses absorbed
microwave energy in the form of heat.
[0072] Macroarray: A panel of a plurality of reaction zones on a
chip ranging from 1 to 1000 zones.
[0073] MATTR: "Microwave-Accelerated Targeted Triggered Reaction"
technology.
[0074] Microarray: A panel of reaction zones on a chip numbering
greater than 1000.
[0075] Microtiter plate: An object commonly used in biomedical
laboratories containing an array of multiple reaction wells.
Typically, microtiter plates are disposable, made of clear acrylic,
and have 24 (arranged in a 4.times.6 array), 96 (8.times.12 array),
384 (16.times.24 array), or 1536 (32.times.48 array) wells.
[0076] Microwave: Electromagnetic radiation in the range of
3.times.10.sup.2 to 3.times.10.sup.4 MHz (wavelengths of 1 m to 1
cm). Dielectric heating occurs in this range, but also occurs at
longer (radio) wavelengths (up to 100 m), which could be
alternatively used. Overall, microwave heating (herein defined to
include radiofrequency dielectric heating) frequencies span
wavelengths of about 1 cm to 100 m.
[0077] Microwave Oven: A device that emits microwave radiation at a
pre-determined wavelength into an internal chamber. The chamber is
typically closed to limit the escape of microwaves.
[0078] Molecular Imprinting: A process whereby specific binding
sites to a chosen target (imprint) molecule are introduced into
synthetic materials. The binding material is usually an organic
polymer. Typically, functional and cross-linking monomers are
co-polymerized in the presence of the imprint molecule, which acts
as a molecular template. Subsequent removal of the template
molecule reveals binding sites that are complementary in shape and
size to the imprint molecule. In this way, molecular memory is
introduced into the polymer, enabling it to re-bind the imprint
molecule with high specificity.
[0079] Nucleic Acid: A large polymer molecule composed of
nucleotide monomers.
[0080] Organic Solution: A liquid medium that is more than 50%
organic solvent by volume.
[0081] Oligonucleotide: A nucleic acid molecule having 99 or fewer
nucleotide residues.
[0082] Polymerase Chain Reaction (PCR): A method for amplifying
specific DNA segments. The method amplifies specific DNA segments
by cycles of template denaturation; primer addition; primer
annealing and replication using thermostable DNA polymerase. The
degree of amplification achieved is a theoretical maximum of
2.sup.N, where N is the number of cycles, e.g. 20 cycles gives a
theoretical 1,048,576-fold amplification (see, U.S. Pat. Nos.
4,683,195 and 4,683,202).
[0083] Polynucleotide: A nucleic acid molecule having more than 99
nucleotide residues.
[0084] Porous: A solid material containing channels through which
water and other liquid molecules can pass.
[0085] RNA: A usually single-stranded nucleic acid similar to DNA
but having ribose sugar rather than deoxyribose sugar and uracil
rather than thymine as one of the pyrimidine bases.
[0086] Thermal Proximity: The situation in which one substance is
close enough to a second substance to permit substantial heat
transfer to occur between them.
[0087] Thermocouple: A sensor for measuring temperature consisting
of two dissimilar metals, joined together at one end. The metals
produce a small unique voltage at a given temperature. The voltage
is measured and interpreted by a thermocouple thermometer.
[0088] Waveguide: A structure that causes a wave to propagate in a
chosen direction. It is accomplished by an intimate connection
between the waves and the currents and charges on the boundaries,
or by some condition of reflection at the boundary.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0089] Directed Microwave Heating
[0090] Dielectric materials are good at absorbing microwaves.
Dielectrics have unique spectral characteristics of frequency
versus heating ability, with different substances heating more
effectively at different frequencies (Gabriel et al., 1998).
Although dielectric heating is referred to here as microwave
heating, dielectric heating can also occur at radio frequencies.
This invention is intended to include those effects.
[0091] Dielectric heating depends on a number of factors including
the frequency of the microwave irradiation and the absorption
properties of the dielectric at that frequency. All dielectric
materials have characteristic absorption spectra (frequency vs.
heating ability). For example, in a conventional kitchen microwave
oven, the microwave frequency (2.45 GHz) is very good for heating
water, but not good for heating other materials (for example, a cup
that holds the water). If the frequency of the microwave emission
would be changed, in theory one could heat the cup but not the
water (depending on the relative dielectric absorption
characteristics of water and the cup).
[0092] In this invention, microwaves heat materials that are
especially good at absorbing microwaves. The microwave-active
materials are in thermal proximity to biological molecules. Heat
from the microwaved materials accelerate reactions associated with
the biological molecules to give a rapid desired result, such as a
biological binding event or a signal indicated the presence of an
unknown biomolecule. Thus, heating of the biological molecules (and
subsequent accelerated chemistry) is targeted by microwave heating
because they are close to the microwave-susceptible material.
[0093] The invention has several advantages over other heating
methods. These alternative methods include IR heating (using a
lamp) and resistive heating. Resistive heating requires direct
contact of the reaction surface with an electrical circuit and
resistor, while the present invention obviates the need for direct
contact. IR heating, although non-contact, is less efficient in
rapidly heating a surface than is microwave heating. Also, some
reactants are photosensitive and damaged by IR light. Finally,
although it is extremely difficult to shine a IR light beam in a
sharp pattern, the present invention allows patterned microwave
absorption by high resolution patterning of microwave-absorbing
dielectrics.
[0094] Physical Components of a Preferred Embodiment of the
Invention
[0095] The physical components of a preferred embodiment of the
invention are:
[0096] 1) The instrument. The instrument (illustrated by reference
to a preferred embodiment shown in FIG. 2) contains; (a) a
microwave source and (b) one or more reaction chamber(s). The
instrument also optionally contains; (c) a means of controlling the
reaction temperature in real time and (d) one or more detection
system(s) to measure physiochemical changes in the sample (e.g.,
light emission and temperature). Each of these will be considered
separately here.
[0097] a. Microwave source. Microwaves can be generated by various
devices including a magnetron, a solid-state device (such as a
Bluetooth or Wi-Fi (IEEE 811.b)), a klystron, a cross-field
amplifier, a traveling wave tube, a backward-wave oscillator, or
any combination thereof. The microwave emission is in the frequency
range of 300 to 30,000 MHz (wavelengths of 1 m to 1 cm). Dielectric
heating also occurs at lower (radio) frequencies of down to 3 MHz
(wavelengths of up to 100 m), which can be alternatively used.
Overall, microwave/dielectric heating frequencies span wavelengths
of about 1 cm to 100 m. Electromagnetic heating throughout this
range is considered part of this invention. The ideal frequency
used depends on factors including the identity of the dielectric
material to be heated. As described above, there are many devices
that generate microwaves--most notable for the present invention
are magnetrons and solid-state devices. Low power magnetrons
(500-1200 W) commonly found in kitchen microwave ovens are
sufficient for the invention. Alternatively, solid-state devices,
such as Bluetooth or Wi-Fi chips, are commonly used in wireless
communication devices. They emit low power (<1 W) microwaves at
the same frequency as kitchen microwave ovens (2.45 GHz). These
devices, which are roughly the size of a house key, are much
smaller than light-bulb-sized magnetrons. Hence, solid-state
devices can generate microwave power in a handheld device. The low
power levels are sufficient for use in this invention, especially
if the dielectric heats well and if the sample to be heated is
placed in a waveguide (see below).
[0098] Attractive frequencies for this invention include 0.915 GHz,
2.45 GHz, 5.85 GHz, and 22.125 GHz. The U.S. Government currently
approves these frequencies for use for industrial, scientific, and
medical uses (Boon & Kok, 1989). Other frequencies may also be
attractive provided that the emission within the microwave chamber
is sufficiently shielded (to prevent interference with
communications uses of microwaves). Of the above-listed
frequencies, 2.45 GHz is attractive because it is a widely accepted
frequency used in numerous existing devices such as domestic
microwave ovens and many wireless communications devices (Wi-Fi and
Bluetooth). Because of widespread use of these devices, design and
manufacturing know-how of 2.45 GHz emitters including magnetrons
and solid-state devices are well known. A frequency of 0.915 GHz is
also an attractive frequency for aqueous applications because water
is least susceptible to dielectric heating at this frequency
(Laslo, 1980).
[0099] b. Reaction chamber. The reactions may be carried out within
an open cavity, such as a microwave oven or a waveguide. Both
microwave ovens and waveguides are well known in the art and
readily adaptable to directed microwave chemistry.
[0100] In the case of an oven cavity, it is preferable for the
microwaves to be "homogenized" to prevent uneven
heating/reactivity. This can be accomplished through the use of a
rotating sample carousel or through the use of irregularities or
deflectors in the oven, which would mix the microwaves.
[0101] A preferable chamber would be a waveguide (for example, sold
by Coleman Microwave Company (Edinburg, Va.) and Gerling Applied
Engineering, Inc., Modesto, Calif.). Microwaves within a waveguide
are very uniform. Moreover, the interior of a waveguide is small
which can be readily used with correspondingly small chips and
plates. One or more holes can be introduced into the waveguide for
practical purposes, such as a slot for plate or chip insertion and
an orifice for light or temperature measurement. Waveguides are
widely available commercially and can also be custom designed based
on known microwave algorithms.
[0102] The dielectric, which is targeted by microwaves, may either
be permanently incorporated into the wall of the reaction chamber
or be a part of the disposable sample support (e.g., a microarray
chip or microtiter plate). In the former case, the sample (a
conventional chip or plate) would be placed on the dielectric in
the reactor chamber. In the later case, the chip or plate would be
modified to include a dielectric layer.
[0103] An alternative reaction chamber to those described above is
one that is outside of a microwave oven or waveguide, yet abuts a
microwave chamber. When this type of reaction chamber is used, the
sample would heat, but not come in direct contact with microwave
irradiation. The sample would be placed in contact with a
dielectric material that is physically built into the wall of the
waveguide/oven cavity. The wall dielectric would heat from the
interior microwave bath, and heat from the dielectric would
thermally transfer to the outside surface where it would contact
the sample. The advantage of this format is that microwaves would
not directly contact the sample to be heated. Thus, materials
incompatible with microwaves could be more easily used. For
example, a metal thermocouple used to measure sample temperature
might spark on the inside of a microwave oven.
[0104] c. Temperature Controller. It is generally desirable to
control the reaction temperature in real time. A thermocouple can
be used to measure the temperature of the dielectric provided that
the dielectric is structurally amenable (for example a chip-based
dielectric. One example is if the dielectric is coated on a
disposable chip (i.e., a microscope slide). A thermocouple could be
used to contact the chip and monitor the temperature during
heating. Moreover, thermocouple temperature measurement could be
used to control the temperature by controlling the power of the
microwave oven. If the dielectric temperature reached a certain
level, say 95.degree. C., the microwave could be automatically shut
off. When the temperature dropped, to say 77.degree. C., the
thermocouple would cause the microwave to begin heating again. Such
thermocouple-based temperature control is well known art (Huhmer
and Landers, 2000; ASTM, 1993; Kreider, 1989). Alternatively,
temperature can be measured using non-contact spectroscopic
techniques (Boon & Kok, 1989; Slyanev et al., 2001). Both
thermocouples and spectroscopic methods have been used to measure
microchip temperatures (Huhmer and Landers, 2000; Slyanev et al.
2001).
[0105] d. Detection System(s). Detection is an attractive (but
non-essential) embodiment of this invention. Detection may be by a
number of means such as fluorescence, absorbance, or
chemiluminescence. In accordance with the principles of preferred
embodiments of the present invention, surface-directed microwave
heating can preferentially enhance numerous chemical reactions,
including reactions that are accompanied by measurable
physicochemical changes, such as chemiluminescence. These
observable reactions are useful in microwave-based molecular
analyses. For analytical applications, the reaction will be chosen
depending on the preferred detection method (a change in color,
luminescence, etc.). The detector is positioned opposing the
reaction, for example on a microtiter plate. It may be within the
reaction chamber, but will preferably not interfere with reaction.
For example, if light emission or transmission is to be measured, a
suitable detector (PMT, CCD camera, human eye, film, or photodiode
array) will be positioned to read signal. In some cases, such as
fluorescence and UV absorbance, the microwave induced
physicochemical change might only be observable upon external
excitation. In these cases, the instrument would require both an
excitation source and a detector. Examples of common excitation
sources are lasers, tungsten lamps, and white light bulbs.
[0106] 2) The Microwave-Absorbing Material. Numerous solid
materials absorb microwaves and consequently heat rapidly. These
materials are either pure or composites with other materials, such
as silicone or plastics. There are many materials that could work
in this invention to absorb microwaves and heat such that the heat
is transferred to accelerate a chemical or biochemical
reaction.
[0107] One material with a high dielectric constant is barium
titanate (BaTiO.sub.3). The dielectric constant is 200-16,000
(compared with 80 for water). Barium titanate can be formed into
films and has been used in analytical devices (Ewart et al, U.S.
Pat. No. 5,922,537). Moreover, in addition to barium titanate,
methods for forming thin and thick films of other ferroelectric
materials at low temperature have improved steadily. Known high
dielectric constant inorganic titanates, niobates, and
ferroelectric polymers can be formed by many processes including
low temperature chemical vapor deposition, laser photo-ablation
deposition, sol-gel processes, RF magnetron sputtering, screen
printing and firing, (in the case of the polymer) spin coating, and
other methods (Yang et al., 1998).
[0108] Natural clay can also be used as a moldable dielectric. In
addition, a 1:1 w/w mixture of alumina-magnetite
(Al2O.sub.3-Fe.sub.3O.su- b.4) can be used as a dielectric support
that heats strongly (Bram et al., 1991). Magnetite
(Fe.sub.3O.sub.4) particles heat well under microwave
irradiation.
[0109] Another material that could be used is carbon. Forms of
carbon include carbon black, activated charcoal, graphite, carbon
nanotubes and nanospheres (such as C.sub.60 and C.sub.70).
[0110] Many additional dielectric materials can be identified by
screening dielectrics for their ability to heat during microwave
irradiation. Class I dielectrics (dielectric constants typically
less than 150) and Class II dielectrics (dielectric constants
typically in the range of 600-18,000) can be used (technical
brochure, Novacap, Inc., Valencia Calif.). Other suitable materials
include organic polymers, aluminum-epoxy composites, and silicon
oxides. The microwave frequency can be varied as well. This simple
screening procedure would yield conditions (frequency and material)
that would direct heating toward the dielectric material.
[0111] Still other materials that heat substantially under RF
irradiation include ferrites and ferroelectrics. In addition to
BaTiO.sub.3, described above, other Perovskites (minerals of the
chemistry ABX.sub.3) such as NaNbO.sub.3, LaCoO.sub.3, LaSrO.sub.3,
LaMnO.sub.3, and LaFeO.sub.3 heat well in a microwave field. Other
materials that heat efficiently in a microwave and which could be
used in the invention include SiC, AlN, ZnO, MgO--SiC,
Al.sub.2O.sub.3, and AlN--SiC.
[0112] Other types of materials that are well known to heat
dramatically under microwave irradiation are various ceramics;
oxides (Al.sub.2O.sub.3, for example), non-oxides (CrB and
Fe.sub.2B, for example), and composites (SiC/SiO.sub.2, for
example) Numerous materials are processed (sintered, etc.) by
exploiting their microwave heating characteristics.
[0113] Microwaves can heat composite materials. For example,
materials that are normally transparent to microwaves can be heated
by adding polar liquids or conducting particles. Refractory oxides
such as alumina, mullite, zircon, MgO, or Si.sub.3N.sub.4 have been
made to couple effectively with microwaves by the addition of
electroconductive particles of SiC, Si, Mg, FeSi, and
Cr.sub.2O.sub.3. Oxides including Al.sub.2O.sub.3, SiO.sub.2, and
MgO have been effectively heated by the addition of lossy materials
such as Fe.sub.3O.sub.4, MnO.sub.2, NiO, and calcium aluminate.
Indium tin oxide (ITO) could also be used. Mixtures of conducting
powders, such as Nb, TaC, SiC, MoSi.sub.2, Cu, and Fe, and
insulators such as ZrO.sub.2, Y.sub.2O.sub.3, and Al.sub.2O.sub.3,
have coupled well with microwaves. Various materials in solution
(zirconium oxynitrate, aluminum nitrate, and yttrium nitrate) that
are good couplers have also been added to enhance microwave
absorption of powdered insulating oxides. A microwave absorbing
heating mantle is sold by Milestone, Inc. made from a composite of
graphitic carbon and Teflon. Microwave-absorbing materials are also
sold by Emerson & Cuming Microwave Products, Inc. (Randolph,
Mass.) . These include ECCOSORB.RTM., which are made from
microwave-absorbing materials (carbon, iron, magnetically, or
ferrite loaded) composited in a polymeric matrix such as silicone,
vinyl or polyurethane. ECCOSORB.RTM. can be purchased in sheets of
various sizes and thicknesses, with or without adhesive
backing.
[0114] Addition of conductive materials in various shapes including
powder, flake, sphere, needle, chip, or fiber, would cause the
heating of low loss materials. For example carbon black or metal
pieces with sizes ranging from 0.1-100 .mu.m can increase the
heating properties when used as inclusions. The nature and
concentration of such materials can be optimized without undue
experimentation (Committee on Microwave Processing of Materials et
al., 1994).
[0115] The microwave-absorbing material can be an integral part of
the microwave-generating instrument, or it can be an accessory
thereof. In this case, the material would be situated in thermal
proximity to the reaction surface. Alternatively, the microwave
absorber can be incorporated into or applied to the bottom of a
disposable reaction vessel such as a microarray chip of 96-well
plate. Numerous application methods are available including
painting (as an ink, such as carbon ink, or in a binder such as
aqueous polyvinyl acetate (PVAc), screen printing (such as SiC in
terpineol), or by adhesive attachment of a polymer composite (such
as ECCOSORB.RTM., Emerson & Cuming)
[0116] 3) The Chemical Reaction. In the broadest terms, the
reaction can be any organic or inorganic reaction that is
accelerated by heat. The reaction will either be: (1) a reaction
that involves biospecific binding or, (2) a reaction that is part
of a sequence of reactions, one of which is a biospecific reaction.
The biospecific reaction typically involves a protein or nucleic
acid molecule (e.g., enzyme, antibody-antigen, and nucleic
acid-hybridization reactions). An example of a sequential reaction
involving a biospecific interaction is enzyme catalysis to form a
product, which then further reacts to form a second product.
Another example of a sequential reaction is peptide synthesis
followed by antibody binding to the synthesized peptide.
[0117] The biospecific binding molecule can be any molecule that is
capable of specifically interacting the reagent(s). The molecule
may be low or high molecule weight, natural or synthetic. Typical
binding molecules could be antibodies, enzymes, receptors, nucleic
acids, molecularly imprinted polymers, and zeolites. These
molecules have specific binding pockets or crevices.
[0118] Reactions run without a bulk solution are useful in
analytical applications (for example in medical diagnostics). In
diagnostics, the reactant solution might contain a biological fluid
from a patient. Microwaves may facilitate the capture and detection
of a molecule of interest.
[0119] The following shows typical directed microwave reactions
covered by this invention:
[0120] i. Preparative Reactions
[0121] 1. Non-specific adsorption (e.g., a protein binding to
plastic)
[0122] 2. Small molecule synthesis (e.g., peptide combinatorial
chemistry)
[0123] ii. Binding Reactions
[0124] 1. Biospecific Protein Binding
[0125] a. Immunoassays
[0126] b. Receptor-Peptide Binding
[0127] 2. Biospecific Nucleic Acid Hybridization
[0128] a. PCR
[0129] b. RNA detection
[0130] iii. Analytical Reactions
[0131] 1. Chemiluminescent
[0132] 2. Fluorescent
[0133] 3. Colorimetric
[0134] iv. Post-Analysis Heat Decontamination
[0135] 4) Formats of Reactions. The support may have any of a
variety of geometries. It may be a planar surface. A suitable
planar dielectric may be part of a chip, such as a multi-analyte
disposable biological assay chip (protein chip or DNA chip) or part
of a microtiter plate. Such apparatus may commonly either possess
the dielectric material as one or more spots on their surface, or
may comprise a continuous layer. Alternatively, the dielectric
material could be in suspension in the form of a particle, such as
a bead or quantum dot. Similar use of dielectric material can be
used in reaction vessels commonly used in biology such as
microarray chips/slides, microtiter plates, test tubes, Petri
dishes, and centrifuge tubes.
[0136] Microtiter plates are in common use to perform biological
analyses (Johnson, 1999). They typically have 96 wells in an
8.times.12 array, but can also have other configurations and
numbers of wells including 24, 384, and 1536. They are (usually)
disposable devices made of acrylic or polycarbonate, but can be
made of essentially any material. The volumes of the wells vary
depending on the number of wells per plate, but 96-well plate wells
hold roughly 150 microliters of liquid. Immunoassays including
ELISAs, enzyme assays, and nucleic acid assays are commonly
performed in wells. Most often, a different assay is performed in
each well (1:1), although it is becoming increasingly common via
low-volume reagent spotting technologies (e.g., Cartesian
Technologies, Inc. (Irvine, Calif.), BioDot, Inc. (Irvine, Calif.))
for multiple assays to be carried out in a single well. Typically,
plates are prepared for analysis by coating the interior surface of
the wells (entirely or by array spotting) with a specific capture
molecule, such as an antibody. Binding usually occurs by
non-specific absorption. Because binding is a surface phenomenon
and bulk heating may denature biomolecules in the aqueous solution
of the wells, it is preferable to target heating to the well
surfaces. This can be done by microwave heating microtiter plates
that have dielectric in thermal proximity to the well surfaces.
This can be accomplished by coating the plate bottoms with
dielectric, for example by painting, or by incorporating dielectric
into the plate material (plastic).
[0137] The effect of mild microwave heating on protein adsorption
in a dielectric coated plate is shown in FIG. 7. The protein avidin
was coated in 96-well plates (Nunc MaxiSorp) under varying
incubation conditions. Plate-bound avidin was then detected using
biotinylated horseradish peroxidase (HRP) as a label to generate
color using the colorimetric HRP substrate, TMB. The height of the
first column in FIG. 7 (Di-elect Coated) represents the relative
amount of protein bound in a microwaved dielectric-coated
(BaTiO.sub.3) plate. The second column (Control 1) shows the
relative amount of protein bound in a non-microwaved
dielectric-coated plate. The third column (Control 3) represents
the relative amount of protein bound in a microwaved
non-dielectric-coated plate. These data show the importance of the
dielectric coating to microwave-accelerated protein binding to
plates.
[0138] Alternatively, conventional plates may be placed in a holder
or mantle that contains dielectric material. In all cases, the
dielectric is in thermal proximity to the surface where biomolecule
attachment occurs. The extent of well coating can be measured by
standard means known in the art, such as total protein or DNA
determination using colorimetric or fluorescent reagents. Coating
can also be analyzed by a function-based assay. Once the well is
coated with the molecule of choice, an analytical reaction can
occur (DNA probe assay, immunoassay).
[0139] There are three preferred ways in which the dielectric
material can be in thermal proximity to the reaction surface.
First, the dielectric can be incorporated into the material that
makes up the solid support. For example, a composite of Teflon and
graphite or barium titanate can be made (Milestone, Inc., Monroe,
Conn.). Microarray chips or microtiter plates can be extruded or
injection molded from the composite. Second, the dielectric can be
attached or deposited as a coating or layer on the solid support.
The dielectric can be painted or spotted to the underside of a chip
or plate (for example, as a barium titanate or carbon paste). It
can also be included within a chip as a "sandwich" layer. Third,
the dielectric can be a solid mantle or holder on/into which the
solid support is placed prior to reaction. In this third format, a
chip or plate can be disposable but the dielectric mantle can be
reused. A dielectric mantle also allows the dielectric material to
have substantial mass (many grams), which facilitates rapid
microwave heating.
[0140] Preferred Methods and Compositions of Matter
[0141] There are numerous ways of practicing the present invention.
Some variables include: altering the microwave frequency and power,
the identity of the microwave susceptible material, the reaction
surface shape (planar or spherical), the reagent capture mechanism
(antibodies, DNA, covalent, non-covalent, etc.), the identity of
the reaction to be accelerated, and practical applications
(analytical, bioanalytical, preparative, etc.). Described below is
a brief overview of some variables and their practical application.
Also described are the current best ways of carrying out the
microwave accelerated targeted reactions.
[0142] One highly attractive format for the invention is to use it
on "chips," i.e., disposable planar surfaces, often made on
microscope slides (for example, 1.times.3 inch rectangles of
glass). Another attractive format is to use it on "microtiter
plates".
[0143] Many attractive potential uses of microwave-targeted
reactions are in the fields of biotechnology/medicine. In these
cases, measured analytes have biological function. Any conventional
assay such as an immunoassay or a DNA probe assay can be carried
out by the described technology. In these assays, well-known
chemical conversions would occur causing a detectable
physicochemical change in some label. For example, chromogenic,
fluorogenic, or luminescent reactions could be carried out.
[0144] A spotting method may be used to deposit reagents. Spotting
(commonly by inkjet printing, pipetting, pin contact or other
high-resolution deposition methods) results in from one to
thousands of reaction spots on the surface of a chip or one to
several (e.g., a 4.times.4 array) spots within an individual
microtiter plate well. There are numerous manual and automated
means of spotting known in the art. Numerous commercial suppliers
exist that sell spotting robots (Biodot, Cartesian) as well as
simple inexpensive devices for spotting (Xenopore). Small volume
analyses on such so-called "microchips" (Schmalzing et al., 2000)
enable huge numbers of assays to be performed on a single chip.
Arrays ("macroarrays" or "microarrays") on chips or in microtiter
plates can be used for analytical purposes. Thousands of assays can
be performed on a single chip or plate using deposition
technologies know in the art, which are commercially widely
available (Pasinetti, 2001; Lennon, 2000; Cooper, 2001; Draghici,
2001; Zubritsky, 2001).
[0145] A preferred way of conducting the procedure is to use
composite dielectric materials containing SiC, carbon Emerson &
Cuming), ferrite (Emerson & Cuming), or barium titanate-based
materials. Carbon may be activated carbon/charcoal (Sigma-Aldrich
Chemical Co.), carbon black (Columbia Chemicals, Marietta, Ga.;
Reade Advanced Materials, Providence, R.I.), graphitized carbon
particles (Polysciences, Inc. Warrington, Pa.). These materials can
be printed or screen-printed onto the reaction device.
Alternatively, dielectric-containing thin or thick films or sheets
can be formed in advance and glued.
[0146] For example, an array of spots can be used to detect genetic
mutations in a myriad of genes. Another example is an immunoassay,
in which an antibody would be present in a spot. Another example is
a ligand assay in which small molecule such as an alkaloid or a
peptide would be present to capture a specific protein receptor.
Chips and plates can be used in numerous analytical applications
including but not limited to; biochemical research, human and
animal medical diagnostics/prognostics, water testing, food
pathogen testing, crop testing, and chemical/biological warfare
agent testing.
[0147] In addition to analytical uses, surface-targeted microwave
heating can be used preparatively. Extending the application of
microchips and microtiter plates described above, the chips and
plates could be prepared for subsequent analytical use using
directed microwave reactions. One general area is the use of
targeted microwave heating to enhance capture protein binding to
plates or chips. Another general area is to use targeted microwave
heating to accelerate on-chip or on-plate synthesis of small
molecule compounds such as peptides or alkaloids. Another is
accelerated biospecific binding involving proteins or nucleic
acids. Another is microwave-targeted PCR.
[0148] In addition to preparative and analytical uses, the
invention can be used for decontamination. If a sample being tested
is pathogenic or toxic, such as an infectious agent or poison,
microwave heating will inactivate the harmful effects. If the heat
generated during analysis is insufficient for destruction,
microwave power/time can be increased to facilitate sample
decontamination.
[0149] Reactions of analytical utility include those that result in
a change in color, luminescence, fluorescence, electrochemistry, or
any other detectable physical property. Preparative reactions
include hydrolysis, peptide or nucleic acid synthesis, and/or
enantioselective reactions, etc. Any preparative reaction is
potentially amenable to the described invention. As with analytical
applications, preparative reactions can be monitored by changes in
a detectable physical property.
[0150] An attractive detection format is chemiluminescence (CL).
These are described in greater detail below in the section
delineating sample practical applications in medicine.
Chemiluminescence reactions can be monitored and quantitated in
many ways including the use of film (for example, X-ray film), or
electronically using a photomultiplier tube (PMT) or a
charge-couple device (CCD) camera. A PMT-based instrument would
involve a microwave-emitting device with a window through which
light is measured. Measurement using a PMT or a CCD camera would be
collected and analyzed using a personal computer and conventional
commercial data acquisition/analysis software (for example,
LabVIEW). Numerous chemiluminescent reactions have been found to be
useful in this invention (FIG. 3).
[0151] As described above, the dielectric material can be in
various formats. Currently the most attractive format is on a chip,
either as spots or as a layer. The use of a "dielectric chip"
allows sensitive detection of multiple analytes. Indeed, microarray
chips or microchips are an attractive application of the
invention.
[0152] The size and features of the instrument will vary with the
practical application. Some feasible types are handheld, portable,
and benchtop.
[0153] Illustrative Preferred Embodiments
[0154] There are numerous practical applications of targeted
triggered microwave reactions. Many are in the fields of analytical
and preparative chemistry. Some though, are in non-analytical
fields. For example, a reaction could be directed at a toxin (such
as a nerve gas) to specifically inactivate that toxin. A capture
step would preferably involve a material such as a molecularly
imprinted polymer. The polymer would trap and concentrate the toxic
gas. Directed microwave heating would then The described invention
could be useful in any practical application where a chemical
reaction is desired and it is important that that reaction is
specific for the chosen molecule.
[0155] Very attractive applications are in the biomedical analysis.
Analyses of biomolecules are critical to diagnostic/prognostic
evaluations. Moreover, scientific research depends on the ability
to detect and measure specific biomolecules. Such biomolecules
include but are not limited to proteins (immunoassay detection) and
nucleic acids (hybridization detection).
[0156] The herein described invention has numerous embodiments and
applications that can be used alone or in series. For example,
preparative, analytical, or decontamination uses could be used
alone or in any combination. Examples include preparative,
analytical, and decontamination embodiments used in series, or
preparative and analytical embodiments, or analytical and
decontamination embodiments. Thus, this invention can be viewed as
modular.
[0157] MATTR Instrumentation in Biomedical Analyses
[0158] There are numerous feasible designs of instruments for
MATTR. In essence, the instrument must be able to bathe the
targeted dielectric in a uniform field of microwave irradiation.
Instruments can be made with one or multiple reaction chambers.
Instruments can also be different sizes, for example, a benchtop
instrument can be made for laboratory use, while a handheld
instrument can be made for field use. A MATTR instrument can be as
small as the smallest microwave emitters combined with the smallest
target reaction chamber. Microwave emitters can be smaller than a
house key (e.g., in cell phones). Target reactions can also be
smaller than a house key (e.g., a small microarray chip).
[0159] Additional features are critical for some applications
including real-time temperature monitoring/control and light
emission measurement capabilities. The basic components of a MATTR
instrument are shown in FIG. 2.
[0160] Experiments described in the Examples below were performed
with a standard kitchen microwave oven. Equivalent or superior
results can be expected using purpose-designed MATTR instruments.
Two such designs are described below for illustrative purposes.
[0161] 1) Waveguide-Based MATTR Instrument.
[0162] a) Microwave Source and Chip Holder (FIGS. 4 & 5).
[0163] The salient features of the system are as follows;
[0164] i. The microwave source is a 1.2 kW magnetron generating
2.450 MHz microwaves.
[0165] ii. The range of microwave power is 0.06-1.2 kW. The
microwave source is controllable both manually and via LabVIEW 4.0
software (National Instruments, Inc.).
[0166] iii. Perhaps the most important feature of the instrument is
that the chips are irradiated in a waveguide, rather than in an
oven cavity. A drawback of microwave ovens is that standing waves
are generated in the oven cavity, resulting in uneven heating
("hotspots"). Hotspots do not occur within a waveguide because
microwaves are uniform.
[0167] iv. The waveguide in the instrument has an opening to give
access to chips. When the chips are inserted, a 13 mm diameter
aperture remains for observation of emitted light. The
configuration of the aperture prohibits escape of microwaves. A PMT
abuts the aperture. The PMT is held in place using a c-mount
adaptor.
[0168] b) PMT Assembly. The PMT assembly is from Applied Scientific
Instrumentation (Eugene, Oreg.). The assembly consists of a
Hamamatsu H5784 series in a custom designed light-tight c-mount
housing. The housing has a special optical collection element to
maximize photon collection. The PMT has a power supply and control
board offering manual and external control of gain.
[0169] c) Infrared Thermometer. Besides measuring light output,
temperature is monitored with a non-contact infrared thermocouple
thermometer (DigiSense Type J thermocouple). The surface
temperatures of chips are read through the light aperture (the PMT
is removed for temperature measurements). Because temperature
readings are not sensitive to visible light, these measurements are
performed manually without the need for light shielding
precautions.
[0170] d) Computer and Software. A personal computer running
LabVIEW 4.0 software is used to simultaneously control the
microwave generator (power and duty mode (on/off timing)) and the
PMT (gain, timing). LabVIEW is used to analyze the collected
data.
[0171] FIGS. 4 and 5 illustrate the above-described microwave
components. FIG. 4 shows the microwave components of a
waveguide-based MATTR instrument. The two boxes in the unit at the
lower left are a power supply and power monitor. The unit at the
upper right is the microwave emission system, including the
microwave source (magnetron, top) and waveguide containing the chip
holder. FIG. 5 gives more details of the microwave components; (1)
magnetron head, (2) switch mode power supply and power monitor, (3)
3-port circulator, (4) short 3 kW dummy load, (5) dual power
monitor leads, (6) precision 3-stub tuner, (7) universal waveguide
applicator, and (8) 13 mm test tube adaptor.
[0172] 2) Oven-Based MATTR Instrument
[0173] The design of an oven-based instrument that uses fiber
optics to detect emitted light is described here;
[0174] a) a suitable microwave oven is made from a microwave
moisture/solids analyzer (model M2, Denver Instrument Co., Arvada,
Colo.). The oven will has a single mode microwave chamber to
provide a uniform power density. The microwave chamber of such an
oven is small and cylindrical and the energy is focused on the
sample. The operating frequency of microwave emission is 2450 MHz.
The output power of the microwave is 550 W and the power source is
115 V, 60 Hz.
[0175] b) The interior of the oven chamber is fitted with a chip
holder that is aligned with the fiber optic cables. The fiber
optics is run from the interior of the microwave to a PMT on the
exterior of the oven. The chip holder supports disposable
dielectric assay chips of various sizes (for example, from
1.times.3 inches up to 5.times.5 inches).
[0176] c) A fiber optic detection system to allow chip imaging
within the microwave chamber. Fiber optics leads from the chip to a
light-recording photomultiplier tube (PMT, Hamamatsu model
H5784-01), which captures light emitted light from the CL reaction,
and
[0177] d) a personal computer that controls and synchronizes the
PMT and the microwave source. The computer runs a versatile data
acquisition, control, analysis, and presentation software package,
(LabVIEW 4.0 software, National Instruments Corp.).
[0178] Reaction Cartridges
[0179] Within a microwave instrument, chemical reactions take place
within reaction vessels, known here as cartridges. Cartridges may
be of any size or dimension, including those commonly used in
chemical laboratories, such as flasks, beakers, vials, and test
tubes. Because the invention inherently involves reactions at or
near surfaces (i.e., near dielectric surface coatings), preferable
cartridges are those with high surface area-to-volume ratios. These
types of cartridges include microtiter plates, microarray chips,
and "lab-on-a-chip" devices. The latter devices are characterized
by small-scale fluidics channels.
[0180] Reaction cartridges have discreet locations for one or more
microwave reactions to be carried out. The cartridges may be
disposable or reusable. The cartridges may contain
microwave-susceptible dielectric. Alternatively, if the dielectric
is permanently incorporated into the instrument reaction chamber,
the cartridges may not have dielectric in their structures.
[0181] Chemiluminescent Compounds for Bioanalytical Assays
[0182] There are very many chemiluminescent reactions known which
efficiently emit light and can be used for bioanalytical purposes.
Some classes of CL reactions are (each of which has many structural
variations); 1,2-dioxetanes, aryl oxalates, acridinium esters,
luminols, and lucigenin. All of these classes have been used
analytically, either as labels in immunoassays or as
chemiluminescent enzyme substrates. In most cases, the
light-emitting chemical reaction that occurs is a bimolecular
reaction, often with an oxidizing agent. Hydrogen peroxide and
sodium hydroxide are common second reagents. All of the reactions
may be accelerated by an increase in temperature. There are vendors
of these compounds such that both free CL compounds as well as CL
compounds labeled with linkers for protein modification for use in
immunoassays.
[0183] Before the present invention of microwave chemiluminescence,
chemiluminescent compounds were considered either glow or flash
type reagents, depending upon whether their reaction rates were
slow (glow) or fast (flash) at ambient temperatures. Reaction rates
and durations were largely beyond the control of the user. With the
current invention, both flash and glow type reagents can also be
considered microwave chemiluminescent reagents. Now both types of
reactions may be controlled by the user using directed microwave
heating. Glow reagents can be caused to flash upon microwaving.
Flash reagents can be rendered unreactive by altering the solvent
conditions and then induced to flash upon microwaving.
[0184] As illustrated in FIG. 3, a diverse variety of
chemiluminescent chemistries can be used as microwave
chemiluminescent reagents. Typical glow type chemiluminescent
reagents are dioxetanes such as compounds 5 and 6 and luminols,
such as 1 and 2. Common flash reagents are acridinium esters. Light
emission by acridan esters (compounds 3 and 4 in FIG. 3) involves
the formation of an acridinium ester intermediate.
[0185] Dioxetanes emit light without any secondary reagent such as
hydrogen peroxide. In addition, dioxetane CL reactions are
remarkably temperature dependent. Dioxetanes are used as glow type
regents in enzyme immunoassays and enzyme assays of alkaline
phosphatase, glucuronidase, glucosidase, and beta-galactosidase
(Tropix, Foster City, Calif.). Dioxetane chemiluminescence is
highly temperature dependent, with light emitted rapidly at
elevated temperatures. Various dioxetanes are commercially
available from Tropix and other sources and methods for conjugating
them to proteins have been published. In addition, Tropix sells
conjugates, which can be linked to proteins.
[0186] Acridinium esters are another class of CL reagents that is
useful in MATTR. These compounds react with acids and bases in the
presence of an oxidizing agent, resulting in flash type CL. Several
acridinium esters are commercially available. Lumigen, Inc.
(Southfield, Mich.) sells small, water-soluble chemiluminescent
labeling acridinium esters that are triggered by a simple chemical
reaction to produce CL as a rapid flash. The chemical kinetics of
these compounds can be slowed by judicious dilution of the
triggering reagents. A flash of light can then be induced by
microwave heating. Another company, Assay Designs, Inc. (Ann Arbor,
Mich.), also sells acridinium ester labeling kits. Their acridinium
esters link to proteins via NHS ester functional groups. Assay
Designs also sells trigger solutions to affect light emission.
[0187] MATTR-Based Assays
[0188] Immunoassays. MATTR-based immunoassays may be conducted in
any of a wide variety of formats. For example, a MATTR chip or
microtiter plate, with specific capture molecules on it surface,
may be exposed to analyte solution, followed by secondary antibody
binding (if necessary), and washing (if necessary) (FIG. 6). An
alternative to testing analytes in solution is a tissue array, in
which immunoassays are performed on thin slices of tissue, usually
arrayed on a chip. Tissue array analyses can also be performed
using this invention.
[0189] Immunoassays are usually performed using either competitive
or sandwich immunoassay formats. The signaling label, generally a
chemiluminescence or fluorescence generating reporter molecule,
will be present on the appropriate surface-bound molecule. Once the
binding and washing has been completed, the chip is placed in the
MATTR instrument and analysis is carried out.
[0190] An example of an immunoassay is one for tumor necrosis
factor alpha (TNF.alpha.). TNF.alpha. is an angiogenic growth
factor protein. There are several commercial sources of high
quality required reagents, TNF.alpha. and appropriate antibody
pairs. R&D Systems (Minneapolis, Minn.) sells a CL-based assay
for this protein that could be used in a MATTR based assay. The
assay is a sandwich enzyme immunoassay. With MATTR, the secondary
antibody is labeled with multiple copies of a chemiluminescent
compound by means known in the art.
[0191] Immunoassays for many different analytes including
TNF.alpha. have been performed using MATTR technology (see
Examples, below).
[0192] Analysis of Nucleic Acids. MATTR-based nucleic acid analysis
will have much in common with immunoassays analysis by the same
technology. The major differences are described here. Assays may
take place as follows;
[0193] (1) A MATTR chip (FIG. 1), with specific capture molecules
on its surface, is exposed to analyte solution and the analyte
binds to the surface. In one type of assay, the captured analyte is
detectable because it itself has been pre-labeled with CL molecules
(Schena et al., 1995). The target cDNA can be labeled using any of
the many well-known methods and reagents (TriLink BioTechnologies,
Inc. San Diego, Calif.; Glen Research Corp., Sterling, Va.). It is
preferable to label the target with multiple CL reporter groups.
For example, DNA can be chemically biotinylated and the biotin-DNA
molecule can bind streptavidin labeled with multiple luminol
molecules. Alternatively a sandwich type format can be employed in
which a secondary probe is used (Kricka, 1999). In this format, the
primary probe, immobilized on the chip, captures the unlabeled
target molecule, which in turn captures the CL-labeled secondary
probe.
[0194] The capture molecule layer is spotted on a nylon membrane.
The nylon may be a full-size overcoat or punched into small
circles. The actual spotting process is carried out using a manual
microarray spotter (Xenopore Corp.), which can deposit spots on a
standard 1".times.3" microscope slide. The manual microarrayer is a
simple bench top device, measuring only 5".times.5" and weighing
less than three pounds. It requires no external power source.
Uncoated or derivatized glass microscope slides, coverslips, porous
membranes, gels, or plastics.
[0195] (2) Once binding has been completed, the chip is placed in a
chip holder in a MATTR instrument and measurement is made. It
should be noted that, as expected, the microwave-generated heat
will denature the analyte, but the signal will not be affected.
[0196] Preparative Applications: Rapid Biomolecular Binding Almost
invariably microtiter plate- or microarray chip-based analyses of
protein or nucleic acids require preparation of the plates or chips
prior to analysis (Price & Newman, 1997; Wild, 2000) involving
coating with a specific protein or nucleic acid. Directed microwave
heating can be used to accelerate these preparations by targeting
heat to proximal dielectrics, which selectively warm the surface of
the plates or chips.
[0197] Plate surface-directed microwave heating gives superior
results when compared to conventional bulk heating in, for example,
a 37.degree. C. incubator chamber. The advantage is related to the
fact that in a bulk incubator the entire plate and its contents are
uniformly warmed while in surface-targeted microwave heating, the
plate binding surfaces are preferentially warmed. Surface binding
reactions are therefore preferentially accelerated. Bulk
(convection) incubators are currently widely used (Harvard
Apparatus, Bellco Glass, Inc., Techne, Inc., Thermo LabSystems,
Inc., Boekel Scientific)
[0198] There are thousands different types of plate or chip
preparative reactions, but typically each involves immobilization
of either a binding macromolecule (protein or nucleic acid) or a
small molecule ligand. Arrays (libraries) of small molecule ligands
such as peptides or other small organic molecules are often
synthesized in situ using combinatorial chemistry methods. The
technology for macromolecule immobilization and small organic
molecule combinatorial chemistry of the present invention is
compared to existing technologies below.
[0199] It is a distinct advantage of the present invention that
surface microwave heating can be used both for preparative
reactions and, in the same location, for subsequent analytical
reactions. Described below are innovative ways in which preparative
and analytical microwave reactions can be used sequentially.
[0200] Preparative Applications: Targeted Microwave PCR
[0201] It is shown here for the first time that directed microwave
heating can dramatically accelerate nucleic acid hybridization.
Polymerase chain reaction is one practical application where
nucleic acid hybridization is essential (McPherson & Moller,
2000; Newton & Graham, 1994). PCR is a nucleic acid
amplification method in which a specific nucleic acid sequence is
geometrically amplified through an iterative process involving
heating and cooling (FIG. 12). In PCR, high temperatures are used
to dissociate strands of double-stranded DNA. Using the invention
described, two steps--annealing (hybridization) and
dissociation--can be accelerated.
[0202] Rapid PCR methods are desirable because of their convenience
and because of the importance of obtaining test results rapidly in
critical lifesaving applications such as biodefense testing (Chen,
2003; Fermer et al, 2003; Gloffke, 2003). PCR hybridization
reactions are slower in the earlier cycles of amplification due to
the low concentration of template (amplified sample) in comparison
to the fairly high concentration of added primer. Conventional PCR
is carried out in a temperature cycling instrument called a thermal
cycler (thermocycler). Thermal cyclers are widely available bulk
heating devices (Constans, 2003). Several designs of thermal
cyclers are available based for example on; heating and cooling by
fluids, heating by electric resistance and cooling by fluid or
refrigerant, heating by electric resistance and cooling by
semiconductors (Peltier devices), and heating matt black metal
surfaces by light followed by air cooling (Newton & Graham,
1994). Thermal cycler-based PCR is relatively slow--an
amplification cycle may commonly take 6 minutes (Example 11).
[0203] Of key importance in this invention is that heating is
non-contact, but bathed in a microwave. There exist infrared
light-heating-based rapid PCR systems (Newton & Graham, 1994;
Giardano et al., 2001; Slyadnev et al., 2001). Systems that use
light as a heat source have the drawback of requiring a direct line
of sight to the PCR reaction. In addition, microwave heating of
dielectrics (and subsequent cooling by heat sinks) requires very
low power, and is extremely rapid.
[0204] The use of microwaves as the heat source in bulk PCR has
been demonstrated (Fermer, 2003). In this publication, binding
(annealing) reactions were not aided by microwave heating--the
reactions were taken out of the microwave during these steps. In
accordance with the principles of the present invention, microwaves
are employed to accelerate nucleic acid annealing. Moreover, in
Fermer et al., the reaction was carried out in bulk solution (0.5
mL plastic tube) without the aid of a proximal solid dielectric--an
added advantage of the current invention.
[0205] Another temperature-dependent aspect of PCR that is commonly
practiced is the sudden introduction of reagents that are entrapped
in hollow wax spheres (Newton & Graham, 1994; McPherson &
Moller, 2000). The idea is that reagents can be physically isolated
by a wax partition until needed. Commercial wax bead products
include Taq Bead.TM. Hot Start Polymerase (enzyme
compartmentalization, Promega), StartaSphere.TM. beads (magnesium
compartmentalization, Stratagene). An alternative is to inactivate
a reagent with an antibody, which heat denatures. JumpStart.TM. Taq
DNA polymerase (Sigma) is an example of this. The DNA polymerase is
temporarily inactivated by a specific antibody, which dissociates
upon heating.
[0206] In the case of MATTR, the wax or other coating agent can be
located in thermal proximity to a microwave-targeted dielectric,
and would melt/dissociate to release the desired reagent upon
microwaving. The transmission of heat from the dielectric to the
coating agent might be especially critical if the coating is made
from a microwave-transparent medium, such as paraffin wax
(Surrmeijer et al., 1990), that would not heat directly by
microwaving.
[0207] It is notable that directed microwave release of an
entrapped or bound reagent from a heat-dissociable compartment is
not restricted to use in PCR. Directed microwave heating can be
used as a general reagent delivery system outside of the field of
PCR. For example, reagents in an immunoassay could be released by
microwaves when needed.
[0208] In the embodiment of rapid PCR, MATTR technology can be used
to rapidly detect mRNA in cancer cells. Detection of mRNA is useful
in determining which of several important cancer-associated
proteins are being produced. Analysis will be performed on cDNA
prepared from cellular mRNA by RT-PCR. RT-PCR is a powerful and
sensitive method for amplifying specific cellular mRNA (Latchman,
1995) and is becoming a powerful method for both qualitative and
quantitative molecular diagnostics (Freeman et al., 1999). In
RT-PCR, mRNA is isolated (either total or polyadenylated RNA). RNA
is then reverse-transcribed to complementary DNA (cDNA) using the
retroviral enzyme, reverse transcriptase ("rt"). Primers (gene
specific or universal) are required to initiate reverse
transcription. Product cDNA is amplified, using PCR or another
amplification method, to give detectable quantities of cDNA. RT-PCR
is an established method that is often used to detect cancer gene
expression (for a review, see Seiden & Sklar, 1996).
[0209] Advantages of the Present Invention in Protein and Nucleic
Acid Immobilization:
[0210] Prior to immunoassays and DNA probe assays, capture
molecules are immobilized on microchips or microtiter plates.
Immobilization is often accomplished by simple adsorption of the
capture molecule to the glass or plastic surface. This procedure
usually involves incubation at room temperature for several hours,
or at 37.degree. for approximately 2 hours. Elevated temperature
heating takes place in a warm chamber (incubator). The present
invention discloses two new and improved ways in which molecules
can be immobilized. The new ways can be used independently or
together. One way is to use microwave heating of the underlying
dielectric to gently incubate the surface at approximately
37.degree. C. Another way is to use the piezoelectric properties of
underlying ceramic dielectrics to create ultrasonic vibration
(sonication), causing the biomolecules to bind to the solid support
faster than without sonication. These two methods are described in
greater detail below.
[0211] (1) Accelerated Immobilization Using Targeted Microwave
Heating. Binding of a macromolecule such as a protein or nucleic
acid is a surface phenomenon. If the surface is warmed, binding
occurs faster. Advantages to microwave surface targeted warming
were surprisingly found. By microwaving the binding surface rather
than the bulk liquid phase as is normally done, heat is directed to
the molecules near the surface. Besides unprecedented capture
molecule binding speed, another advantage of using dielectric
surface heating is that the same dielectric can be used later for
analytical purposes. Protein or DNA capture molecules are bound
using microwaves, and then microwaves are used to accelerate a
reaction (chemiluminescence, for example) in analyte detection. It
has been found that (as is documented in standard protocols)
incubating at elevated temperatures such as 37.degree. C.
accelerates both non-specific attachment of proteins to surfaces
and biospecific (e.g., antibody-antigen) binding on solid phases.
Both types of binding can be accelerated by directed microwave
surface heating as described herein.
[0212] Advantages of the Invention in Solid Phase Combinatorial
Chemistry:
[0213] Increasingly large libraries of low molecular weight
compounds are screened or tested for the ability of proteins or
nucleic acids to bind to them. The present invention surprisingly
can be used both for synthesis (on chip or plate or other solid
support) and for subsequent analysis. First, directed microwave
heating is used to accelerated chemical synthesis on the surface,
then microwave heating is used in the analysis of the surface
(e.g., chemiluminescent receptor binding assay or immunoassay).
Advantages of small-scale synthesis, followed by analysis include
the fact that less waste is generated (an environmentally-sensiti-
ve method) and microwave heating on a small scale uses less energy
(microwave heating is in general considered a "green
technology").
[0214] Solid phase-immobilized libraries of chemical compounds can
be synthesized (Dolle, 2000) and screened for activity against
biomolecules. For example, peptide libraries can be made to search
for lead compounds that bind to a protein receptor. Reports of
microwave-assisted combinatorial chemistry have been published
(Kappe, 2001; Borman, 2001) and combinatorial chemistry on chips
has also been reported (Fodor et al., 1993; Kramer &
Schneider-Mergener, 1998; MacBeath et al, 1999). Microwave-assisted
combinatorial synthesis on standard (non-dielectric-coated) planar
surfaces has been reported (Blackwell, 2003), but not on
dielectric-coated surfaces. The advantages associated with
dielectric coating are unique to this invention. These include;
speedy heating under low microwave power, the ability to direct
heat in a pattern on the planar surface, and the ability to use
microwaves in post-analysis, for example using directed microwave
chemiluminescence.
[0215] Smaller libraries (2-1000 distinct chemicals) called focused
libraries can be made. The libraries can then be assayed on the
same chip using microwave-accelerated reactions to detect binding
or catalysis.
[0216] Bulk (as opposed to directed) microwave accelerated
chemistry has existed in the literature for almost twenty years and
is becoming increasingly popular with organic chemists involved in
combinatorial chemistry and drug discovery (Lew et al., 2002;
Wathey et al. 2002; Kappe, 2002). To fill the growing demand, at
least three companies; Personal Chemistry (Uppsala, Sweden), CEM
(Wilson, N.C.), and Milestone (Italy) offer instruments and kits
for solution phase reactions. All commercial products involve
solution phase syntheses in irradiated tubes. Although not yet
significantly commercialized, solid phase, solvent-free microwave
chemistry is also becoming increasingly visible in the scientific
literature (Wathey et al., 2002). Microwave chemistry often reduces
reaction times by 10-fold or more, while increasing yields
substantially.
[0217] On-chip dielectric-directed microwave combinatorial
chemistry (MATTR) has not been reported in the before this
invention. In MATTR, the reaction mixture is indirectly targeted by
microwaves--the dielectric chip is the targeted and heat transfer
to the proximal reactants drives the reactions. Evaporation is
minimized by an inert chip cover, if necessary. In essence, MATTR
for the first time unites the fields of microarrays, combinatorial
chemistry, and microwave chemistry. On-chip libraries can be
prepared using spots on a contiguous chip surface (e.g.,
nitrocellulose membrane) or by using surface membrane-entrapped
beads. Functionalized beads are commonly used in conventional
combinatorial chemistry.
[0218] Until now, on-chip synthesis has been unattractive because
in many cases the size of the effort was not worth the information
gained in an experiment. Conventional (long) reaction times were
necessary to prepare chips that would be used only once or twice,
and then discarded. MATTR will drastically reduce the synthesis
time so that synthesis and analysis can be done in a single day.
Moreover, MATTR is a low-solvent volume chemistry that generates
little waste.
[0219] Directed chemistry is described above as the small-scale
synthesis of chemical libraries on, for example chips or plates,
followed by bioanalysis for binding to a molecule such as a
protein. However, there is no reason to preclude the use of
directed microwave synthesis in larger scale synthesis. Larger
quantities, milligrams or grams, of a compound could be synthesized
as well. Thus, attractive medicinal qualities of a molecule could
be discovered by on-chip synthesis and detection, and rapid
directed microwave synthesis could then be used to scale up
production for further detailed studies.
[0220] Use of MATTR To Decontaminate Samples After Analysis.
[0221] MATTR-based analyses could often involves testing of toxic
or pathogenic specimens. For example, in the field of biodefense,
tests for toxins such as the protein ricin, or pathogens such as
the bacterium anthrax could be carried out. These samples would
need to be decontaminated after analysis. Using MATTR, samples
could be decontaminated using microwave heating. In
decontamination, a MATTR chip, plate, or other disposable cartridge
would be heated to inactivate or denature the pathogen or toxin.
For example, a bacterium could be inactivated by heating in excess
of 160.degree. C. for 5 minutes. A proteinaceous or nucleic acid
toxin could similarly be denatured. Thus, a MATTR-based analysis
can encompass one or more aspects of microwave-accelerated
preparation, analysis, and decontamination.
[0222] Having now generally described the invention, the same will
be more readily understood through reference to the following
Example, which is provided by way of illustration, and not intended
to be limiting of the present invention.
EXAMPLE 1
[0223] Microwave Detection of Luminol and Isoluminol
[0224] Luminol (3-aminophthalhydrazine) and isoluminol
(4-aminophthalhydrazine) (FIG. 3, compounds 1 and 2, respectively)
are two chemiluminescent compounds that are commonly used in
bioanalyses. Normally, they are made to emit light; through the
action of an enzyme such as horseradish peroxidase (HRP), in a
non-enzymatic ambient temperature chemical reaction, or through an
electrochemical reaction. (Iso)luminol have not been reported to
emit light upon directed microwave heating. It is not obvious that
rapid microwave heating would cause (iso)luminol to emit
light--rapid heating could destabilize the molecules, resulting in
a breakdown by non-light-producing pathway(s). Moreover, in
accordance with the principles of preferred embodiments of the
invention, conditions are sought in which little or no
chemiluminescent reaction occurs at room temperature, but an
extremely fast reaction (approaching a "flash") occurs under mild
microwave heating.
[0225] Luminol and isoluminol rapidly react with oxidants at
alkaline pH to produce light at room temperature. Numerous
conditions are tested at room temperature and under microwave
heating. Variations included buffer (sodium bicarbonate, sodium
phosphate, Taps, imidazole, Hepes, Bes), pH (6.5, 7.5, 8.39, 8.55,
8.71, 9.10), oxidants (hydrogen peroxide, sodium perborate,
potassium permanganate, iodine), and enhancers (copper sulfate,
4-iodophenol, cobalt chloride).
[0226] Room temperature reaction rates were measured at various
concentrations of reactants in 96-well plates. Plates were read in
a luminometer (Thermo Labsystems Ascent).
[0227] Microwave heating reaction rates were tested on FAST Slides
(S&S Biosciences, Keene, N.H.), which are nitrocellulose-coated
microscope slides, and on HydroGel Slides (PerkinElmer), which are
hydrogel-coated microscope slides. Both types of slides were
undercoated with microwave-active dielectric material (BSR-1 SS6M,
0.01", Emerson & Cuming, Randolph, Mass.). Luminol (Alfa Aesar,
Ward Hill, Mass. and Alpha Innotech, San Leandro, Calif.) and
isoluminol (Sigma, St. Louis, Mo.) were spotted on the slides using
a hand microarrayer (S&S MicroCaster), which deposited 3-5 nL
spots. Combined remaining reagents were sprayed to uniformly wet
the slide. The slides were placed in a clear plastic protective
holder and X-ray film (ECL Hyperfilm, Amersham) was placed against
the holder facing the reactants. Light was recorded on the film
either at room temperature or during microwave heating (for
generally 30 sec) in a GE microwave oven (600 W, Model JE635 WW
06).
[0228] Throughout the experiment, light emission yield from luminol
(was consistently higher than from isoluminol. Isoluminol and
luminol responded similarly to changes in reaction conditions,
indicating that the chemical mechanisms of the two compounds were
essentially the same. The spray reagent found to be most suitable
for directed microwave detection of luminol and isoluminol
included; 67.5 (or 125 mM) mM NaBO.sub.3, 100 mM NaHCO.sub.3 (pH
7.6), and 0.0175 g/L CoCl.sub.2. These conditions gave a very slow
room temperature chemiluminescence reaction, but a very fast
reaction under mild microwave heating (approx. 100.degree. C.). The
reaction rate ratio (microwave/room temp.) was estimated to be
greater than 1000.
[0229] The detection limit for both FAST- and HydroGel-based
dielectric slides was 853 amol of luminol. Isoluminol showed a
detection limit 8-10 times lower than luminol.
EXAMPLE 2
[0230] Microwave Detection in an Immunoassay Using
Isoluminol-Labeled Antibody
[0231] Example 1 showed that small amounts of (iso)luminol (2, FIG.
3)could be detected using microwave directed chemiluminescence. An
experiment was carried out to see if microwave directed
chemiluminescence could be used to detect an analyte in an
immunoassay. Another purpose of the experiment was to see if a
direct-labeled (isoluminol) antibody could be used.
[0232] In the experiment, mouse IgG (Sigma I-5381) was used as the
analyte. Mouse IgG (1 mg/mL) was spotted (3-5 nL) on a chip (FAST
Slide) in S&S Array Buffer. Analyte was detected using
streptavidin-isoluminol (3.5 labels per streptavidin, Sigma S-8532)
that was bound to the recognition antibody, which was a biotin goat
anti-mouse antibody (Sigma B-7151). Chemiluminescence of chips
(BSR-1 dielectric undercoated FAST Slides) was measured on X-ray
film following spraying with reagent (see Example 1) at room
temperature or upon microwaving.
[0233] Detection was performed on BSR-1/SS6M or MCS/SS6M dielectric
(Emerson & Cuming) undercoated slides--either FAST Slides
(S&S) or homemade nitrocellulose coated microscope slides.
Homemade slides are made by spraying microscope slides with Elmer's
spray adhesive and placing Protran nitrocellulose (S&S) on the
sticky slide.
[0234] Initially, streptavidin-isoluminol was spotted and detected
at various concentrations and found to have a microwave-directed
chemiluminescence detection limit of approximately 22.7 fmol of
streptavidin.
[0235] For detection of mouse IgG, the following protocol was
followed. Analyte spotted chips were blocked with S&S
Wash/Block buffer for 2 hours at room temperature on a rocker.
Chips were then washed 3 times for 5-10 minutes with the same
buffer. The pre-formed complex of [biotinylated goat anti-mouse
antibody][streptavidin-isoluminol] was then added and the chip was
incubated at room temperature on a rocker for 5 hours. The chip was
then washed as described above.
[0236] The chips were sprayed with the reagent described in Example
1, and microwaved for 30 seconds. Microwave chemiluminescence light
emission was captured on X-ray film. Spots corresponding to the
analyte spots were visible on the scanned film, indicating that;
(1) microwave chemiluminescence immunoassays can be performed, and
(2) sensitive detection of a direct label (unamplified signal) is
possible.
EXAMPLE 3
[0237] Microwave Detection of Chemiluminescent Enzyme Substrates
Having discovered that (iso)luminol can be used in microwave
chemiluminescence analyses, experiments were conducted to determine
if other chemiluminescent chemistries were also amenable to
microwave triggering. Chemiluminescent compounds can often be used
as direct labels (attached to proteins or nucleic acids) or enzyme
substrates. Streptavidin-isoluminol is an example of the former and
luminol (an HRP substrate) is an example of the latter. Enzyme
substrate chemiluminescent compounds have the advantage of
catalytic amplification, but they also have the drawback of light
emission being a sustained low glow. Chemiluminescent enzyme
substrates usually follow the pathway shown below;
E+S.fwdarw.E+P*.fwdarw.E+P+hv
[0238] where E is enzyme, S is substrate, P* is a transient
product, P is final product, and hv is light emission. Metastable
product P* spontaneously breaks down to form P and light. Thus, if
enzyme catalysis is slower than the conversion of P* to P, then a
glow directly corresponds to the time course of enzyme catalysis.
If enzyme turnover is faster than the conversion of P* to P, then
P* accumulates and slowly breaks down in a glow. In either case,
glow chemiluminescence can be very time consuming (hours) and often
results in interfering background signal, which builds up with
time. The goal was to convert the conventional pathway (above) into
one where little or no glow occurs, but instead a microwave-induced
flash;
E+S.fwdarw.E+P*(metastable).fwdarw.microwave.fwdarw.E+P+hv.
[0239] Numerous chemiluminescent enzyme substrates with diverse
chemistries are commercially available. The goal was to test a
representative range of substrates and develop as many as possible
as microwave substrates. The compounds tested were; APS-5 (3 in
FIG. 3, Lumigen, Inc., Southfield, Mich.), PS-3 (4 in FIG. 3,
Lumigen), Lumi-Phos Plus (LPP, 5 in FIG. 3,_Lumigen), and CDP-Star
with Nitroblock II (6 in FIG. 3, Tropix/Applied Biosystems,
Bedford, Mass.). Of these, APS-5, LPP, and CDP-Star are sold as
alkaline phosphatase (AP) substrates, and PS-3 is sold as a
horseradish peroxidase (HRP) substrate.
[0240] Although these are enzyme substrates, they were first tested
them with microwave heating in the absence of enzymes to determine
whether they might have chemistries that cause them to emit light
under directed heating.
[0241] 1.0-2.0 .mu.L of substrates (APS-5, PS-3, LPP, CDP-Star, all
supplied as liquids) were therefore spotted on dielectric chips
(BSR-1/SS6M undercoated, Protran nitrocellulose coated microscope
slides), and read light emission (30 or 60 seconds, room
temperature or microwave) on X-ray film (Amersham Hyperfilm ECL).
One of the compounds, APS-5, was detected at room temperature, and
the light emission intensity became substantially greater when
microwaved.
[0242] The experiment was then repeated the experiment, but the
chips were sprayed with oxidant solution before detecting light
emission (see Example 1 for spray reagent). Here, both APS-5 and
PS-3 emitted light at room temperature and much more light under
microwave heating. Light emission from APS-5 appeared greater than
the light emission in the absence of oxidant.
[0243] The goal was to modify the reagent spray in order to lower
the room temperature reactions, but not the microwave reactions, of
APS-5 and PS-3. Various solutions including sodium bicarbonate (0.2
M, pH 9.6 and 7.5) and oxidant spray with cobalt concentration
varied were tried without success. It was found that using the
spray oxidant listed in Example 1, but replacing sodium bicarbonate
with equimolar amount of Tris buffer, pH 9.3 gave a very good
result for APS-5. The microwave/room temperature light emission
ratio was estimated to be 100 and the APS-5 detection limit was
approx. 1 fmol.
[0244] The goal was to identify conditions whereby PS-3 would emit
little light at room temperature but much light under mild
microwave heating. the effect of the compositions of the Tris, pH
9.3 reactant spray described above were explored by varying pH
(10.1, 11.1, 12.0), varying the concentration of NaBO.sub.3, and
comparing NaBO.sub.3 to H.sub.2O.sub.2 as the oxidant. It was found
that the best conditions for PS-3 were the same as for APS-5 (Tris
solution), but with a pH of 11.1.
[0245] In conclusion, APS-5 and PS-3, but not LPP or CDP-Star, were
found to emit light in the absence of enzyme. In both cases,
chemical triggers gave substantially more light under microwave
heating.
EXAMPLE 4
[0246] Enzyme-Amplified Microwave Detection of Chemiluminescent
Compounds
[0247] The results of Example 3 showed that APS-5 and PS-3
chemiluminescence can be detected in the absence of enzymes and
that the intensity of this non-enzymatic chemiluminescence is
greatly enhanced by directed microwave heating. Next, experiments
were conducted to determine if enzyme-catalyzed luminol, APS-5,
LPP, PS-3 and CDP-Star could be adapted to directed
microwave-enhanced detection. Because these substrates are designed
to emit light upon enzymatic catalysis, it was important to find
conditions whereby enzyme catalysis (S.fwdarw.P* in Example 3) and
light emission (P*.fwdarw.P+hv) could be aecoupled--in other words,
conditions in which catalytic conversion of substrate to metastable
product is rapid, but subsequent conversion to final product (P)
and light was minimal. The second step (P*.fwdarw.P+hv) would then
be triggered by directed microwave heating. If this were possible,
then microwaves would trigger a sudden burst of light from the
accumulated metastable product.
[0248] luminol and PS-3 were investigated with the enzyme
horseradish peroxidase (HRP) and APS-5, CDP-Star, and LPP with the
enzymes acid phosphatase (AcP) and alkaline phosphatase (AP). In
this Example, it is shown that for all five enzyme substrates
listed above, reaction conditions were found under which light
output at room temperature was minimized and rapidly restored by
microwave heating.
[0249] Experiment 1: Horseradish Peroxidase (from horseradish,
Sigma P-8125) with luminol and PS-3. Each substrate was looked at
individually.
[0250] HRP and luminol. In the absence of enzyme, luminol reacts at
alkaline pH with oxidants such as H.sub.2O.sub.2 and NaBO.sub.3.
This reaction is accelerated by HRP. The natural pH optimum of HRP
catalysis is around 6.0-6.5, but the optimum pH of luminol light
emission is at pH 9 or greater. It was anticipated that by running
the HRP-luminol reaction at near neutral pH, the enzyme reaction
would proceed at a rapid rate, but the subsequent light emitting
reaction would be slow or inert, until triggered by microwave
heating. 0.5 .mu.L each of luminol +/- HRP (50 ng) +/-
H.sub.2O.sub.2 (10 mM) were spotted on Protran
nitrocellulose-coated, BSR-1/SS6M dielectric-undercoated slides.
Some of the slides were sprayed with the spray reagent described in
Example 1 except that the buffer and pH were modified. Three
minutes after spraying, the light emission at room temperature or
under microwave irradiation for 10, 30, or 60 seconds was measured.
X-ray film (ECL Hyperfilm) was used to detect and measure light
emission. The buffers tested were Tris at pH 6.6, 7.0, 7.1, 7.2,
7.7, and 9.3. The results showed a pH dependence of light
emission--the trend showed more light was given off at higher pH
values. Substantial light was given off at room temperature.
Microwave heating enhanced light emission, but only by 2-fold or
less. These data suggest that enzyme catalysis and light emission
are strongly coupled in the luminol/HRP reaction.
[0251] HRP and PS-3. It was found that one could decouple HRP
catalysis and light emission in this reaction. Under these
conditions, HRP catalyzed PS-3, but very little light was emitted
until the dielectric chip was microwaved.
[0252] HRP, PS-3, and H.sub.2O.sub.2 (0.5 .mu.L each) was spotted
on Protran nitrocellulose, BSR-1 dielectric chips, incubated at
room temperature for 3 minutes, then read at room temperature or
under microwave heating for 30 seconds. HRP was diluted into
various buffers including pH 9.3 (50 mM Tris), pH 6.5 (50 mM BES),
pH 7.5 (50 mM HEPES). The results with HEPES, pH 7.5 buffer were
spectacular--little or no light emission occurred at room
temperature, but upon microwaving very dark spots were seen on the
X-ray film. No spots were seen if HRP was left out. This indicates
that a metastable intermediate (P* above) accumulates and that the
intermediate breaks down under microwave heating, but not at room
temperature.
[0253] Experiments were also conducted with HRP, PS-3, and
NaBO.sub.3 (sodium perborate tetrahydrate, Aldrich 244120), in the
complete absence of H.sub.2O.sub.2. Perborate buffers gave better
light output when used as an oxidant in place of peroxide. The best
substrate/buffer solutions for microwave-enhanced chemiluminescence
were found to include 5-25 mM perborate, 100 mM PIPES buffer, pH
7.0, and 50% (v/v) PS-3 substrate. These buffers gave high enzyme
activity, low light output at room temperature, and high light
output under microwave heating.
[0254] Experiments were also conducted to investigate the use of
perborate-containing buffers in the HRP/PS-3 detection system.
These included pH studies and inclusion of cobalt ion. Ultimately,
it was found that the best substrate/buffer for the HRP/PS-3
microwave detection system was 50% (v/v) PS-3 (Lumigen), 40%
phosphate-citrate (50 mM) buffer with sodium perborate (0.03%), pH
5.0 (Sigma P-4922), and 10% (v/v) 250 mM sodium perborate. The use
of 10% (v/v) 125 mM perborate instead of 250 mM gave similarly
successful results.
[0255] Experiment 2: Alkaline Phosphatase (bovine intestinal
mucosa, Sigma P-6774) with CDP-Star, LPP, and APS-5. Each substrate
was looked at individually.
[0256] CDP-Star (Applied Biosystems, supplied as 0.25 mM
"ready-to-use" with Nitroblock II) was mixed with nothing
("straight"), or mixed 1:1 with water (control), Tris (pH 8.0, 250
mM), glycine (pH 9.0, 250 mM, or NaHCO.sub.3 (pH 10.0, 250 mM).
Substrate was mixed 3:1 with enzyme (approx. 1 u/.mu.L) and the
mixtures were spotted (0.2 .mu.L) on a Protran-coated, BSR-1/SS6M
dielectric undercoated slide. X-ray film measurements at times
between 0 and 30 minutes of incubation (room temperature and
microwave readings) showed a dramatic effect of microwave heating,
especially with Tris buffer, and less with bicarbonate buffer. Room
temperature readings were faint while microwave readings were
dark.
[0257] Lumi-Phos Plus (LPP, Lumigen, Inc. supplied ready to use)
was tested as described above for CDP-Star. LPP gave similar
results to CDP-Star--there was little light emission at room
temperature, but dramatic light emission under brief microwave
irradiation. The darkest spots on film were from straight LPP
(undiluted). The other spots all gave substantial emission and
there was no strong pH or buffer effect.
[0258] APS-5 (Lumigen, supplied ready to use) also showed
microwave-enhanced light emission when tested in the same manner as
CDP-Star and LPP. However, this substrate shows quite a bit more
background emission than the other substrates. Non-enzymatic room
temperature light emission (see above) was observed, as well as
much more enzyme-catalyzed light emission without microwave
triggering.
[0259] In conclusion, all three alkaline phosphatase substrates
tested showed microwave-enhanced luminescence. CDP-Star and LPP
showed little background luminescence in the presence of enzyme
until microwaved, indicating that metastable intermediate (P* in
Example 3) was accumulating. APS-5 showed relatively high
non-enzymatic and enzymatic room temperature emission suggesting
that P* was much less stable than it is in CDP-Star and LPP.
[0260] Experiment 3: Acid Phosphatase and CDP-Star, LPP, and APS-5.
Acid Phosphatase (Type IV-S from potato, Sigma P-1149) was tested
as an enzyme signal amplifier. This enzyme is optimally active at
pH 5-7. If signaling reactions are run at the optimum pH of this
enzyme, the enzyme-catalyzed portion of the reaction would be fast
(S.fwdarw.P*), but the optimally-alkaline light emission step might
be slow (P*.fwdarw.P+light), until induced by sudden microwave
heating.
[0261] The experiment was carried out as described above for
alkaline phosphatase, except buffers used were; sodium acetate (pH
5.0, 250 mM), imidazole (pH 6.0, 250 mM), and sodium phosphate (pH
7.0, 250 mM). Enzyme/substrate incubations were varied from 0-30
minutes on chips prior to X-ray film reading at room temperature or
under microwave heating.
[0262] The results showed that CDP-Star is a substrate of acid
phosphatase at pH 5.0. Spots could only be seen on film when the
chips were microwaved, but not at room temperature. CDP-Star
appears to be a much better substrate of alkaline phosphatase than
acid phosphatase. No enzyme activity could be detected with either
LPP or APS-5 under any conditions tested.
EXAMPLE 5
[0263] Microwave Detection of Chemiluminescent Compounds on
Chips
[0264] In accordance with the principles of preferred embodiments
of the present invention, molecules on chips can be detected by
microwaving the chip and detecting emitted light using X-ray film
facing the chip. Alternatives to this method were discovered during
this work.
[0265] In one alternative, the invention provides a method in which
the chip on which the molecules would retain function following
microwave analysis. In most cases, the chips had been used once,
and then discarded. The heat (generally 90-100.degree. C.) is
believed to denature the biological reagents (antibodies, etc.) on
the chip. A "blot" method, in which the substrate is not directly
applied to the original chip, but instead wetted a glass slide
covered with a layer of nitrocellulose and undercoated with
BSR-1/SS6M dielectric was investigated. This second chip was
pressed against the original chip for a period of time
corresponding to the assay time (usually 2-3 minutes). The second
chip was then removed from the original chip and assayed for
enzymatic product. Assays were performed either by room temperature
or microwave chemiluminescence.
[0266] Various membranes were successfully used including; AE-100
nitrocellulose (Schleicher & Schuell), Unisart CN200
nitrocellulose (Sartorius), GFF Conjugate pad (glass fiber,
Millipore), SNHF 04000 slow flow lateral membrane (Millipore), and
Protran nitrocellulose (Schleicher & Schuell). The use of
Immobilon P PVDF (Millipore) was investigated, but this membrane
was found to wet poorly. To generate light, HRP, PS-3, and
H.sub.2O.sub.2 was spotted on the original chip, blotted, and then
the second chip was read. Results showed that dark well-defined
light emission spots were seen on the secondary chip, indicating
that the product of PS-3 remained on the second membrane after
catalysis. This experiment demonstrated that spots on the original
chip could be non-invasively assayed by directed microwave
heating.
[0267] A variation of the above experiment was performed. In this
"overlay" method, the second chip had no dielectric undercoating.
When wetted with substrate, the second chip was overlaid onto the
original chip. It was found that when the membrane was wet,
the-second chip (and the glass slide backing) were essentially
transparent to light. Thus, the second chip could be overlaid on
the original chip and light could be detected through the back of
the second chip. This format is attractive, because the second chip
provides protection from the film (or camera or PMT). Experiments
showed that the "overlay" method gave sharply defined dark spots on
film corresponding to enzyme-catalyzed, microwave-triggered
chemiluminescence.
EXAMPLE 6
[0268] Microwave Chemiluminescence Detection in an Immunoassay
[0269] Chip-based immunoassays were performed to see if
enzyme-amplified microwave-enhanced chemiluminescence could be used
as a detection method.
[0270] Experiment 1: An assay for mouse IgG protein (the analyte)
was carried out as follows. BSR-1/SS6M undercoated FAST slides
(nitrocellulose, Schleicher & Schuell) were used as chips.
Mouse IgG (Sigma I-5381, reagent grade) was dissolved to 0.5 mg/mL
in Schleicher & Schuell Array Buffer and spotted on chips,
either by pipetting (0.2 .mu.L) or by spotting with a microarrayer
(S&S MicroCaster). After arraying, the chips were covered with
S&S hybridization chambers and blocked with S&S Wash/Block
Buffer for 2 hours on a rocker. The buffer was replaced with
biotinylated goat anti-mouse antibody (Sigma B-7151, Fab specific)
at concentrations of either 0.5 .mu.g/mL or 100 ng/mL, again in
S&S Wash/Block Buffer. The chips were allowed to incubate for
90 minutes on a rocker then washed twice with S&S Wash/Block
Buffer, then washed twice with PIPES buffer (83 mM, pH 7.00). The
detection enzyme, streptavidin-horseradish peroxidase (SA-HRP,
Amersham RPN1231 diluted 35,000:1), was then bound to the
biotinylated complex as recommended for 45 minutes. The chips were
washed with S&S Wash/Block buffer (2.times.10 min.) and PIPES
buffer (3.times.10 min.).
[0271] Chips were overlaid with a layer of S&S AE100
nitrocellulose membrane (which becomes transparent when wet) and
wetted with oxidizing buffer (1:1 mix of PS-3 substrate (Lumigen)
and 25.0 mM NaBO.sub.3, 100 mM PIPES, pH 7.0). Chips were incubated
for 2-minutes at room temperature. Subsequently, light emission was
read for 30 seconds, either at room temperature or under microwave
irradiation (600 W). Light emission was read on Hyperfilm ECL
(Amersham) film.
[0272] Results showed that one could see very dark spots
corresponding to analyte on chips. Spots were very dark, so the
oxidant concentration was lowered to 0.5 and 5.0 mM NaBO.sub.3,
with 5.0 mM giving better results. Although one could see spots in
control experiments (room temperature), the spots were
significantly darker under microwave irradiation.
[0273] Experiment 2: An immunoassay for mouse IgG was performed on
HydroGel.TM. Slides (PerkinElmer). HydroGel slides are glass slides
coated with a thin layer of clear hydrogel for protein analysis.
Slides were prepared and used essentially according to the vendor's
instructions. Mouse IgG (the analyte, Sigma Chem. Co., Cat. #
I-5381) was spotted at 0.5 mg/mL using a MicroCaster Arrayer
(S&S), which gives spots of 3-5 nL volume. Chips were blocked
using S&S Wash/Block Buffer then incubated with goat anti-mouse
IgG antibody (biotinylated FAB-specific, Sigma B-7151) at
concentrations ranging from 0.01-1.0 .mu.g/mL in the same buffer.
After washing, the chips were incubated with Amersham
Streptavidin-HRP (Cat. RPN 1231), then washed again. Chips were
undercoated with BSR-1 dielectric for reading. Chemiluminescent
reagent was the same as described above in Experiment 1. Chips were
read generally with a 2 minute room temperature incubation to allow
enzyme catalysis, followed by equal-time room temperature or
microwave analysis on X-ray film. Microwaving (600 W, 2450 Hz) was
generally for 30 seconds. The results showed faint light emission
at room temperature analysis but very dark spots under microwave
analysis. These results show that microwave chemiluminescence is
superior to stand chemiluminescence on HydroGel.TM. chips.
EXAMPLE 7
[0274] Microwave Chemiluminescence Detection of Cytokines and IgG
Numerous experiments were performed to see if microwave
chemiluminescence could be used to detect a diverse range of
analytes in several immunoassay formats. The analytes included the
human cytokines tumor necrosis factor alpha (TNF-.alpha.),
interferon gamma (IFN-.gamma.), and interleukin 1 beta
(IL-1.beta.). The formats included chips that had assay surfaces
that were coated with hydrogel (PerkinElmer), nitrocellulose
membranes (Panomics, Inc.), and nitrocellulose casts (S&S). In
different experiments, capture antibodies were variously arrayed by
hand spotting (using a pipettor or hand microarrayer), mass
commercial spotting, and custom-commercial spotting.
[0275] Experiment 1. Schleicher & Schuell Provision Slides
contain a pre-spotted array of 16 different anti-cytokine capture
antibodies spotted on cast nitrocellulose. This commercially
available chip is used to monitor and measure human cytokines. It
is sold in kit form with reagents for immunoassay steps and also
comes with instructions for conducting an on-chip immunoassay for a
panel of cytokines.
[0276] Two cytokines were measured, separately and together
-IFN.gamma. (human recombinant, R&D Systems Cat#285-IF) and
TNF.alpha. (human recombinant, R&D Systems Part#840121). The
analytes were measured at a range of concentrations, 128 fg/mL-1
ng/mL TNF.alpha. and 192 fg/mL-3ng/mL IFN.gamma.. The assay matrix
was S&S Wash/Block Buffer. The Provision manufacturer's
instructions were essentially followed using the kit components to
form sandwich immunoassay complexes on the chips.
[0277] For detection, chips were undercoated with Emerson &
Cuming BSR-1 dielectric. Detection reagent, consisting of 50% PS-3
(Lumigen), 40% citrate/phosphate/perborate (Sigma), and 10% 250 mM
sodium perborate, was applied to the chip and dabbed to remove
excess liquid. Following a 2-minute room temperature incubation to
allow enzyme catalysis, the chip was microwaved (600 W, 2450 MHz)
for 30 seconds. Light was captured on X-ray film for a total of 60
seconds (during 30 seconds of microwaving plus an immediate
additional 30 seconds).
[0278] Scanned X-ray films showed that the cytokines could be
detected down to 1.6 pg/ml TNF.alpha. (possibly 68 fg/mL was
borderline-visible) and 2.4 pg/mL IFN-.gamma. (96 fg/mL was
borderline-visible). In a control chip with no dielectric backing,
not even the highest TNF.alpha. concentration of 1 ng/mL could be
detected. This experiment was shown to be reproducible.
[0279] Experiment 2: TNF.alpha. detection on nitrocellulose-coated
FAST Slides (S&S) was performed using microwave
chemiluminescence detection. The manufacturer makes FAST Slides
using a casting process to coat glass slides with porous
nitrocellulose.
[0280] Human TNF.alpha. and antibodies for detection were obtained
from R&D Systems (DuoSet ELISA Development System, Cat. #
DY210). The kit contained capture antibody, detection antibody,
TNF.alpha. standard, and streptavidin-HRP. Standard protocols were
used for washing and incubation. Capture antibody was spotted on
slides using a hand pipette. Incubations were carried out with
rocking using chip hybridization chambers (S&S). Analyte
(TNF.alpha.) was used at 2.0 ng/mL.
[0281] Assays were performed on six chips. Chips were analyzed at
room temperature with no microwaves and by microwave
chemiluminescence (essentially as in Experiment 1 above). Chips
were undercoated with BSR-1 dielectric. The results on X-ray film
showed that microwave chemiluminescence resulted in dark spots on
the X-ray films corresponding to the chip locations of TNF.alpha.
capture antibody but no spots could be seen in the standard
chemiluminescence analysis, either by eye or following digital
scanning. These data show that microwave chemiluminescence gives
much greater signal than corresponding conventional
chemiluminescence on FAST Slides.
[0282] Experiment 3: Microwave Chemiluminescence detection on
Panomics membrane arrays was also tested (TranSignal.TM. Human
Cytokine Antibody Array 1.0). Panomics nitrocellulose membranes are
unsupported nitrocellulose pre-spotted with antibodies for an array
of cytokines. Capture antibodies for 18 cytokines are spotted in
duplicate. The membranes also have positive and negative control
spots. Human TNF.alpha. (2.0 ng/mL, R&D Systems) was used as
the analyte. The immunoassay protocol was followed according to
instructions, except for the detection. Panomics recommends
conventional chemiluminescent detection of cytokines on membranes.
Two membranes were assayed and detected using the chemiluminescent
reagent described above in Experiment 1. The membranes were placed
on BSR-1 undercoated microscope slides for analysis. The membranes
were measured at room temperature after a 1-5 minute incubation to
allow enzyme catalysis and under microwave conditions (600 W, 2450
Hz, 30-45 sec.). Spots on non-microwave membranes were barely
visible on X-ray film, but microwave chemiluminescence spots were
sharp and dark. The positive controls and TNF.alpha. spots were
very dark, but no signal was seen from any of the other cytokine
spots. This demonstrates that microwave chemiluminescence is
superior to standard chemiluminescence using Panomics membrane
antibody arrays.
[0283] Experiment 4: An immunoassay for the cytokines TNF.alpha.
and IL-1.beta. was performed using FAST.TM. Slides that were custom
spotted by Schleicher & Schuell. Slides were purchased that
were pre-spotted with 1 mg/mL TNF.alpha. and IL-1.beta. capture
antibodies, each in a 3.times.3 array (18 spots total). Equimolar
cytokine mixtures were assayed on five chips. Each chip was used to
detect a different cytokine concentration; 0.05 ng/mL, 0.1 ng/mL,
0.5 ng/mL, 1.0 ng/mL, and 5.0 ng/mL. Analyte solutions of human
TNF.alpha. (R&D Systems, Cat# 210-TA) and human IL-1.beta.
(R&D Systems, Cat# 201-LB) were made up in S&S Wash/Block
Buffer. Chips were each enclosed in a hybridization chamber and
blocking, washing, and binding steps were carried out essentially
as recommended by S&S for FAST Slides using S&S Wash/Block
Buffer. Biotinylated secondary antibodies and streptavidin-HRP were
from R&D Systems (anti-TNF.alpha. was from DuoSet kit, and
anti-IL-1.beta. was Cat# BAF201, and streptavidin-HRP (diluted
200:1) was Part 890803). Chips were undercoated with BSR-1
dielectric for reading. Chemiluminescent reagent was the same as
described above in Experiment 1. Chips were read generally with a 2
minute room temperature incubation to allow enzyme catalysis,
followed by equal-time room temperature or microwave analysis on
X-ray film. Microwaving (600 W, 2450 Hz) was for 15-30 seconds. The
results showed that the lowest concentration of tested cytokines
(0.05 ng/mL) could be detected with microwave chemiluminescence and
individual points could be resolved, even though they were closely
spaced (1.6 mm between spots). Both cytokines were visible with
IL-1.beta. giving a slightly greater emission. These results show
that microwave chemiluminescence is superior to stand
chemiluminescence on custom pre-spotted chips, that both IL-1.beta.
and TNF.alpha. could be detected at 0.05 ng/mL, and that micrometer
spot-to-spot resolution is possible.
EXAMPLE 8
[0284] Microwave-Accelerated Antibody-Antigen Binding
[0285] The invention demonstrates surface-targeted mild microwave
heating causes acceleration in the rate of biospecific protein
binding. This is described here for binding of goat antibody
binding to mouse IgG (the antigen), and for mouse antibodies
binding to human TNF.alpha. and IL-1.beta..
[0286] In the first case (biotinylated goat antibody binding to
mouse IgG), approximately 4 nL of 0.5 mg/ml mouse IgG in PBS was
spotted on FAST slides (S&S) and allowed to air dry for 3
hours. The slides (or "chips") had been undercoated with
adhesive-backed BSR-1 dielectric (0.01 inch thick, Emerson &
Cuming, Randolph, Mass.). The chips were washed 3 times with
S&S Wash/Block buffer (S&S, Keene, N.H.), and then blocked
for 1 hour in the same. One set (4) chips was subjected to mild
microwave incubation with goat anti-mouse antibody (1 .mu.g/ml)
while the other set (4) of chips was subjected to room temperature
incubation (same concentration of antibody). Room temperature
incubation (24-27.degree. C.) was for 60 or 120 minutes. Microwave
incubation was for 10 minutes, in the range of 30-49.degree. C.
Following incubation, the chips were again washed in S&S
Wash/Block buffer. The chips were then incubated for 45 minutes in
streptavidin-HRP (Amersham) that had been diluted 500:1 in PBS. The
chips were then washed in S&S Wash/block buffer.
[0287] The chips were read using microwave chemiluminescence. The
substrate mixture included five parts Lumigen P-3 substrate, four
parts Sigma citrate/phosphate/perborate buffer, pH 5.0 (Cat
#P-4922), and 1 part 250 mM sodium perborate (Aldrich Cat.
#244120). The chips were incubated for 2.0 minutes to allow for
enzyme catalysis, and then microwaved for 30 seconds. Emitted light
was read on Hyperfilm (Amersham) autoradiography film. Only one of
the four room temperature incubated showed binding of antibody to
antigen. However, the chips that had been mildly microwaved for 10
minutes showed bold spots where antibody bound to analyte. These
results showed that directed mild microwave heating is far superior
to room temperature incubation in allowing antibody to bind to
antigen.
[0288] In the second case, it was found that specific mouse
antibodies bind much more rapidly to the cytokines TNF.alpha. and
IL-1.beta. when mildly microwaved than when incubated
conventionally at room temperature. In these experiments,
"sandwich" immunoassays on FAST slides (S&S) that had been
pre-spotted with a capture antibodies specific for each of the two
cytokines (S&S Biosciences, Keene, N.H.). Adhesive undercoats
of BSR-1 dielectric (Emerson & Cuming) were applied.
Immunoassays were performed using cytokines and secondary
antibodies from R&D Systems (Minneapolis Minnesota). Three
types of immunoassays were performed: (1) standard room temperature
assay (2-hour capture antibody-antigen incubation and 2-hour
secondary antibody-antigen incubation), (2) short room temperature
assay (as above except using two 10 minute incubations), and (3)
short microwave assay (same as above except both incubations were
10 minutes under mild microwaving). These three types of assays
were performed in triplicate (three chips each). The chips were
then blocked and washed as described above.
[0289] Following formation of the antibody-analyte-antibody
sandwich, HRP reporter was bound to label HRP. Streptavidin-HRP
(Amersham) bound to the biotinylated secondary antibodies on the
chip as recommended by the manufacturer. As above, detection was by
microwave PS-3 chemiluminescence.
[0290] Detection on X-ray film (Hyperfilm ECL) showed that,
consistent with the results above, microwave incubation
dramatically accelerated binding rates. One could easily detect
both cytokines with microwave incubations and standard room
temperature incubations, but could barely see the analytes with
short room temperature incubations (FIG. 8).
EXAMPLE 9
[0291] Microwave Chemiluminescence Detection of DNA
[0292] Experiments were performed an experiment to see if one could
use microwave chemiluminescence to detect nucleic acids, such as
DNA. The goal of the experiment was to detect a linear 4 kb plasmid
containing a 1 kb insert of the mouse glyceraldehydes 3-phosphate
dehydrogenase (GAPDH) gene. A modification of the method described
in Stillman & Tonkinson (2000) was employed.
[0293] In an Eppendorf tube, DNA was reacted with psoralen-biotin
(BrightStar, Ambion, Inc., Austin, Tex.) according to
manufacturer's directions. The labeled DNA (1 ng/mL) was then
spotted on S&S FAST Slides at volumes ranging from 0.1-1.0
.mu.L. After drying (about 1 hour), the chips were covered with
hybridization chambers (S&S) and were blocked with S&S
Wash/Block Buffer for 2 hours on an orbital rocker. The chips were
then washed three times in PBS/0.1% Tween buffer (10 min/wash) and
two times in PBS buffer (10 min/wash). After washing,
streptavidin-HRP (Amersham) was added at dilutions varying from
5000:1 to 35,000:1 and rocked for 1 hour. The chips were then
washed as above, but also with two quick (1 min) washes in 83 mM
PIPES buffer, pH 7.0. HRP-labeled DNA was detected using PS-3
chemiluminescent substrate (Lumigen) modified for microwave
chemiluminescence. An equal volume of substrate (PS-3) was mixed
with either pH 5.0 citrate/phosphate/perborate (as in Example 8) or
pH 7.0 PIPES/perborate. Slide-sized nitrocellulose membranes
(AE100, S&S) were then wetted with substrate-containing buffer
and overlaid the DNA chips with the wet, transparent membranes.
After 2-3 minute incubations to allow for enzyme catalysis, the
membrane-covered chips were microwaved and light emission (through
the overlaid AE100 membrane) on X-ray film was detected.
[0294] Light emission was observed upon microwave irradiation (30
sec detection) but not in similar room temperature detection. This
demonstrates that microwave chemiluminescence can be used to detect
DNA on chips. It is estimated that 379 attomoles of DNA was
detected using microwave chemiluminescence.
EXAMPLE 10
[0295] Microwave-Accelerated Nucleic Acid Hybridization
[0296] Experiments were performed to test whether mild microwave
irradiation can accelerate nucleic acid-nucleic acid hybridization.
Nucleic acid hybridization is an important step in DNA and RNA
testing in plates and microarray chips.
[0297] A plate-based mRNA quantitation kit from R&D Systems
(Quantikine mRNA, Cat. RN000, Minneapolis, Minn.) was used. The
assay kit includes all reagents except for specific detection
probes and analytes ("targets"). Probes and calibrator for human
.beta.-actin (Genbank Accession 3NM001101.2, cDNA=866 bp, Cat#
RN188)) and human COX-2 (Genbank Accession NM.sub.--000963,
cDNA=4465 bp, Cat# RN171-036) were purchased from R&D Systems.
In the experiments, .beta.-actin mRNA was used as the analyte
(target) and COX-2 mRNA as the control.
[0298] The assay format involved a "sandwich" type hybridization
where the target mRNA was hybridized at both ends to gene-specific
probes. One probe is biotinylated to allow capture on a solid phase
streptavidin-coated 96-well plate. The other probe is labeled with
digoxigenin to allow binding of the reporter-generating molecule.
The reporter-generating molecule is a conjugate of an
anti-digoxigenin antibody and alkaline phosphatase (AP). The kit is
sold for colorimetric detection, but hybridized mRNA was detected
using a luminescent AP substrate (CDP-Star with Nitroblock II,
Applied Biosystems, Bedford, Mass.).
[0299] Hybridization was performed in four plates ( )--two plates
were incubated in a 65.degree. C. water bath and two plates were
undercoated with Emerson & Cuming BSR-1 dielectric and mildly
microwaved. The specific procedure was as follows.
[0300] First, .beta.-actin probes were mixed with a range of
calibrators (0-400 amol/mL COX-2 and 0-150 amol/mL .beta.-actin) in
four 96-well plates (Costar 3590) using kit buffers according to
manufacturer instructions. Two of the plates (for microwaving) were
undercoated with Emerson & Cuming BSR-1 dielectric (0.01"
thick). Each plate was incubated separately; (1) in a 65.degree. C.
water bath for 60 minutes, (2) in a 65.degree. C. water bath for 10
minutes, (3) 10 minute microwave (600 W intermittent,), and (4) 5
minute microwave (600 W intermittent). For microwave incubations,
the plate temperature held at approximately 55.degree. C. Following
incubations solution was transferred to wells of a
streptavidin-coated plate (part of kit) and allowed binding of
biotinylated nucleic acid. Following binding, anti-digoxigenin-AP
conjugate was washed and added, and allowed to bind. After
additional washes, CDP-Star substrate was added to the wells. After
a suitable 5 minute incubation, the chemiluminescence was read in a
standard luminometer (Luminoskan Ascent, Thermo LabSystems).
[0301] Results showed that microwave incubation (55.degree. C.) is
quicker than water bath incubation (65.degree. C.) (FIG. 11).
Controls (COX-2 or no mRNA) gave little or no signal, but -actin
mRNA gave strong signals. Signals were not seen with 5-minute
microwave incubation, but this was believed to be an artifact.
These results indicate that mild microwaving provides at least
six-fold faster hybridization of nucleic acids than waterbath
incubation.
[0302] This experiment was repeated (10 minute microwave and 60
minute waterbath) with essentially the same result.
EXAMPLE 11
[0303] Microwave-Accelerated PCR
[0304] Directed microwave heating can be used to accelerate DNA
amplification by PCR (Newton & Graham, 1997; McPherson &
Moller, 2000). The sample of DNA to be amplified and PCR reagents
are placed in a dielectric-coated vessel (such as a PCR tube) or on
a dielectric-coated chip (such as described in Giordano et al.,
2001). Other formats are possible as long as they have a reaction
surface that is in thermal contact with a microwave-targeted
dielectric. DNA can be immobilized on a surface solid phase or in
solution proximal to the dielectric. Results described in Example
10 demonstrated that nucleic acid hybridization occurs faster in a
dielectric-coated vessel than in a vessel at the same (or higher)
temperatures. Thus, the dielectric coating is essential for the
invention.
[0305] DNA can be amplified in a SiC dielectric-coated PCR tube.
The tube is prepared by mixing SiC powder (400 grit) with Elmer's
Glue-All (aqueous-based PVAc). The mixture is painted on the
surface of the tube and allowed to dry.
[0306] The DNA to be amplified can be a sequence of choice but for
example can be a 500-base-pair-fragment of DNA from .gamma. phage
(Giordano et al., 2001). Appropriate oligos are prepared.
[0307] The procedure that is followed is similar to that described
as Protocol 2.1 in McPherson & Moller (2000) except that a
microwave oven (600 W, 2450 MHz) is used instead of a thermocycler
to vary the temperature and the cycle times are different. All
reagents and other equipment are as described. Below is shown the
recommended thermocycler temperatures and time regime for a cycle
of amplification:
[0308] (a) 94.degree. for 5 min (to denature the template);
[0309] (b) 94.degree. for 1 min;
[0310] (c) 55.degree. for 1 min;
[0311] (d) 72.degree. for 1 min; (repeat (b)-(d) 25-40 times;
[0312] (e) 72.degree. for 2 min (to ensure all molecules are
completely synthesized).
[0313] Reagent temperatures under microwave conditions are held
essentially the same. Temperatures can be monitored using an IR
optical pyrometer. Microwave incubation times are 10-fold shorter.
Minor optimization of the microwave protocol may be required which
will involve shortening incubation times 10-fold at each individual
temperature, while holding the other incubation times for the other
temperatures as listed above. For example, steps (a)-(b) (at
94.degree.) are shortened from 360 seconds to 36 seconds while the
other steps are held as shown above. DNA yields are measured by
conventional means. Steps that that can be shortened without
substantially impairing the final yield of amplified DNA are
shortened in the final optimized microwave protocol.
EXAMPLE 12
[0314] Microwave-Accelerated Tissue Microarray Analysis
[0315] Tissue microarrays are thin sections of normal or diseased
human, animal, or plant issue generally placed on microscope slides
or cards (Simon et al., 2004; Dutton 2003). The presence and
distribution of molecular species on the arrays can be probed with
specific antibodies or single stranded nucleic acids. Biospecific
binding of a probe to a tissue microarray obeys the same
physicochemical principles as binding to a molecular microarray
chip. Mild microwave irradiation can accelerate biospecific
molecular binding to a tissue array. In addition, detection can be
carried out using microwave chemiluminescence.
[0316] Tissue microarrays can be made by the user or purchased from
a vendor. IMGENEX Corp. (San Diego, Calif.) is a major vendor of
ready-made tissue array slides, brand named Histo.TM.-Array Slides.
Microwave incubation and detection of human TNF.alpha. can be
performed on Histo.TM.-Array Slides; such as formalin-fixed human
tumor tissue slides (e.g., Cat. #'s IMH-301, IMH-304, IMH-306).
[0317] The manufacturer's (IMGENEX) instructions are followed
except where for directed microwave incubation and detection steps.
Prior to being used in directed microwave incubation/detection, the
slides are undercoated with BSR-1 dielectric. Binding of the
primary antibody (1 .mu.g/mL biotinylated mouse anti-human
TNF.alpha. monoclonal antibody, BD Pharmingen, Cat# 554551) is
accelerated by intermittent microwaving (600 W, 2450 MHz) for 10
minutes at 35-45.degree. C. This is 12-fold shorter time than the
manufacturer (IMGENEX) recommended binding time of 120 minutes at
room temperature. Following binding, the slides are washed and
incubated with streptavidin-HRP (500:1 dilution, Amersham).
Detection of the enzyme HRP (and hence TNF.alpha.) is carried out
by microwave chemiluminescence using the formula (Lumigen PS-3)and
method described above.
[0318] Similarly, in situ hybridization (detection of specific RNA)
can be carried out on tissue microarrays. Again, the IMGENEX
protocol is followed except for rapid microwave-accelerated
hybridization and microwave chemiluminescence. The probe
hybridization step is carried out on a dielectric
(BSR-l)-undercoated slide for 60 minutes in a microwave oven (600
W, 2450 MHz, slide maintained at 45-55.degree. C.). This is 16-fold
shorter than the vendor recommended time of 16 hours in a
50.degree. C. incubator.
EXAMPLE 13
[0319] Microwave-Accelerated Cytokine Analysis Using Fluorescence
Detection
[0320] Microwave-accelerated binding was compared to room
temperature binding of nine cytokines to capture antibodies on
nitrocellulose chips. This experiment was carried out using a
Schleicher & Schuell FASTQuant kit, which can be used to
analyze the presence and concentrations of human IL-10, IL-13,
IFN.gamma., IL-1.beta., IL-2, IL-4, IL-5, IL-6, and TNF.alpha.. The
chips are sold with pre-printed specific capture antibodies for
these cytokines printed on each slide.
[0321] Each chip has 16 assay pads, each consisting of a 6.times.6
antibody array. The arrangement of capture antibodies within each
assay pad is shown below:
1 1 2 3 4 5 6 1 positive positive positive IL-6 IL-6 IL-6 control
control control 2 IL-1.beta. IL-1.beta. IL-1.beta. IL-10 IL-10
IL-10 3 IL-2 IL-2 IL-2 IL-13 IL-13 IL-13 4 IL-4 IL-4 IL-4
IFN.gamma. IFN.gamma. IFN.gamma. 5 IL-5 IL-5 IL-5 TNF.alpha.
TNF.alpha. TNF.alpha. 6 positive positive positive positive
positive positive control control control control control
control
[0322] The reaction pads on a chip can be spatially partitioned
S&S 16-well chambers, thus allowing each 6.times.6 array to be
individually assayed. Four chips were assayed, each with eight
assay zones used. One each chip standard curves consisting of eight
cytokine concentrations were tested. The eight zones had the
following cytokine cocktails:
2 IL-1.beta., IL-2, IL- IL-10, IL-13, 4, IL-5, IL-6, Reaction
IFN.gamma. TFN.alpha. Zone (pg/mL) (pg/mL) 1 50,000 10,000 2 12,500
2,500 3 3,125 625 4 781.25 156.25 5 195.31 39 6 48.83 9.76 7 12.2
2.44 8 0 0
[0323] Two chips were assayed according to manufacturer's
instructions (and using included kit reagents and buffers) with one
exception. The other two chips were also assayed according to the
same instructions, but with one exception. Instead of the
recommended room temperature capture of cytokines on capture
antibodies for 3 hours, chips were undercoated with Emerson &
Cuming BSR-1 dielectric (0.01" thick) and intermittently microwaved
for 15 minutes. Microwaving (600 W GE microwave) was monitored
using an infrared thermometer and the chip temperatures were
maintained between 30 and 42.degree. C. To maintain this range,
microwaves were pulsed with a sequence of roughly approximated 3
seconds on and 90 seconds off.
[0324] All secondary antibodies (included in kit) were labeled with
streptavidin-Cy.sup.5 (Amersham Biosciences) as recommended by
S&S and chips were scanned with a GenePix fluorescence
microarray scanner (Axon Instruments, Inc., Union City Calif.).
[0325] Results showed that a 15-minute microwave incubation gave
brighter spots (with minimal background) than a 3 hour incubation
at room temperature. FIG. 9 shows digitized scans from a room
temperature chip (R1-R4) and a microwave chip (M1-M4). The numbers
1-4 refer to the reaction zones (see above).
[0326] These results are important in that they show the
universality of accelerated microwave binding--all nine cytokines
bound better under directed microwave binding than at room
temperature. All of the analytes have different molecular
structures and unique binding specificities. The results also show
that fluorescence monitoring can be used.
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[0393] All publications and patents mentioned in this specification
are herein incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually indicated to be incorporated by reference in the
entirety.
[0394] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
* * * * *