U.S. patent application number 12/819830 was filed with the patent office on 2011-01-20 for biocatalytic solgel microarrays.
This patent application is currently assigned to Rensselaer Polytechnic Institute. Invention is credited to Douglas S. Clark, Jonathan S. Dordick.
Application Number | 20110015088 12/819830 |
Document ID | / |
Family ID | 23314326 |
Filed Date | 2011-01-20 |
United States Patent
Application |
20110015088 |
Kind Code |
A1 |
Dordick; Jonathan S. ; et
al. |
January 20, 2011 |
BIOCATALYTIC SOLGEL MICROARRAYS
Abstract
A system and method for conducting high-throughput interactions
between test compositions and analytes, comprising one or more test
compositions, and a plurality of independent micromatrices, wherein
each said micromatrix encapsulates at least one said test
composition; and said micromatrices are made of a material that is
permeable to an analyte.
Inventors: |
Dordick; Jonathan S.;
(Schenectady, NY) ; Clark; Douglas S.; (Orinda,
CA) |
Correspondence
Address: |
ELMORE PATENT LAW GROUP, PC
515 Groton Road, Unit 1R
Westford
MA
01886
US
|
Assignee: |
Rensselaer Polytechnic
Institute
Troy
NY
The Regents of the University of California
Oakland
CA
|
Family ID: |
23314326 |
Appl. No.: |
12/819830 |
Filed: |
June 21, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11598306 |
Nov 13, 2006 |
7846747 |
|
|
12819830 |
|
|
|
|
10287442 |
Nov 1, 2002 |
7267958 |
|
|
11598306 |
|
|
|
|
60336045 |
Nov 1, 2001 |
|
|
|
Current U.S.
Class: |
506/9 ; 506/14;
506/18; 506/7 |
Current CPC
Class: |
B01L 2300/0829 20130101;
B01L 2300/069 20130101; B01J 2219/00364 20130101; B01J 2219/00596
20130101; B01J 2219/00725 20130101; B01J 2219/00835 20130101; B01J
2219/0072 20130101; B01L 3/5085 20130101; B01J 2219/00585 20130101;
B01J 2219/00837 20130101; B01L 2300/0819 20130101; B01J 2219/0086
20130101; B01J 2219/00527 20130101; B01L 3/5027 20130101; B01J
2219/00691 20130101; B01J 2219/00317 20130101; B01J 19/0046
20130101; B01J 2219/00497 20130101; B01J 2219/00743 20130101; B01J
2219/00533 20130101; B01J 2219/00644 20130101; B01J 2219/00536
20130101; B01J 2219/00722 20130101; B01J 19/0093 20130101 |
Class at
Publication: |
506/9 ; 506/7;
506/14; 506/18 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 30/00 20060101 C40B030/00; C40B 40/02 20060101
C40B040/02; C40B 40/10 20060101 C40B040/10 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by grant
number BES-9902878 from the National Science Foundation. The
Government has certain rights in the invention.
Claims
1. A method for high-throughput screening to detect a reaction or
reaction product having a desired feature, comprising the steps of:
a. providing an apparatus, said apparatus comprising a plurality of
independent, permeable micromatrices fixed on a solid support,
wherein each said micromatrix encapsulates at least one test
composition and wherein the test composition is selected from the
group consisting of an enzyme, a cofactor, an antibody, a cell
extract, a cell fragment and a cell; b. combining one or more
distinct applied compositions with said micromatrices under
conditions suitable for reacting said applied compositions with
said test compositions, wherein each applied composition contains a
xenobiotic; and c. assaying each reaction or reaction product in
step (b) for a desired feature.
2. The method of claim 1, wherein the test composition comprises a
constituent of mammalian origin.
3. The method of claim 2, wherein the test composition comprises a
constituent of human origin.
4. The method of claim 1, wherein the test composition is a
cell.
5. The method of claim 1, wherein the test composition comprises a
cytochrome P450 isoform.
6. The method of claim 1, wherein the micromatrices are fixed in a
well, channel, conduit or depression; or fixed on a raised
platform; or surrounded partially or totally by a raised wall or
barrier; or surrounded partially or totally by a depressed channel;
or a combination thereof.
7. The method of claim 6, wherein the micromatrices are surrounded
partially or totally by a depressed channel.
8. The method of claim 6, wherein the micromatrices are fixed on a
raised platform.
9. The method of claim 1, wherein a distinct applied composition is
combined with a distinct test composition.
10. The method of claim 9, wherein numerous distinct applied
compositions can be combined with numerous distinct test
compositions in parallel.
11. The method of claim 1, wherein the applied composition is added
to the apparatus by dipping the apparatus into a solution of
applied composition.
12. The method of claim 1, wherein the product of a reaction is
combined with a second test composition.
13. The method of claim 1, wherein each said micromatrix is
comprised of the same material, wherein said material comprises a
solgel, a hydrogel, a polyacrylamide, a polyacrylate, a polyvinyl
alcohol, a polyvinylene, or a polyvinyl silicate, and wherein said
material is substituted or unsubstituted.
14. The method of claim 13, wherein the micromatrices are combined
with an applied composition by submerging the micromatrices in a
solution comprising the applied composition.
15. The method of claim 1, wherein the micromatrices are
impermeable to the test composition.
16. The method of claim 1, wherein the method is
high-throughput.
17. An apparatus comprising a plurality of independent, permeable
micromatrices fixed on a solid support, wherein each said
micromatrix encapsulates at least one test composition, wherein the
test composition is selected from the group consisting of an
enzyme, a cofactor, an antibody, a cell extract, a cell fragment
and a cell and wherein the micromatrices are fixed on a raised
platform.
18. The apparatus of claim 17, wherein the test composition
comprises a constituent of mammalian origin.
19. The apparatus of claim 17, wherein the test composition
comprises a constituent of human origin.
20. The apparatus of claim of claim 17, wherein the test
composition is a cell.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/598,306, filed Nov. 13, 2006, which is a divisional of U.S.
application Ser. No. 10/287,442, filed Nov. 1, 2002 (now U.S. Pat.
No. 7,267,958), which claims the benefit of U.S. Provisional
Application No. 60/336,045, filed on Nov. 1, 2001. The entire
teachings of the above applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Chemicals affect living organisms in both positive and
negative ways. A new drug can save lives, or an environmental
contaminant can create health problems. Sometimes, the same
chemical can have both positive and negative effects, such as a
drug that cures a disease but also has side effects. Multiple
chemicals can interact to produce unexpected effects, for example
when some medications taken in combination lead to side effects.
For example, terfenadine (SELDANE.RTM.) was removed from the market
in 1998 because its interaction with other drugs resulted in fatal
heart arrhythmias. One study in the U.S. attributed as many as
100,000 deaths per year in the U.S. to such adverse drug reactions
(ADR), making it between the 4.sup.th and 6.sup.th leading cause of
death.
[0004] Chemicals can have different effects on different organisms,
for example, potential drugs that work in animal studies, but later
fail in human trials. Chemical effects also differ between
individuals. Many medications only help a percentage of patients
because patients respond to drugs in different ways. Chemicals
effects also vary between body tissues. For example, some
environmental toxins affect specific organs like the liver or the
brain.
[0005] A major reason for these differences is that species,
individuals, and organs all have different kinds and amounts of
enzymes. Enzymes are part of the machinery of living cells that
allow cells to react to drugs and to break down chemicals. In
humans, a large group of enzymes in the liver are responsible for
the majority of drug interactions and side effects. Different
levels of these enzymes are responsible for many of the variations
in the effects of chemicals.
[0006] There is a need for a technology to rapidly, effectively,
and economically test the health effects of chemicals. Such
chemicals include potential new life-saving pharmaceuticals,
environmental contaminants, workplace toxins, potential
carcinogens, and beneficial food chemicals, among many others.
Current methods either involve testing on live animals, which can
be time-consuming and costly, or involve testing in the laboratory,
which is often not relevant to human health.
[0007] At the same time, there is a need for a technology to speed
up the drug development process. One major bottleneck in the race
to develop new life-saving treatments is the optimization of new
drug candidates. When a potential new drug is discovered, teams of
chemists often modify its chemical structure to create new
compounds, and then screen them for improved efficacy and reduced
side effects. This process currently is extremely expensive,
intricate, time-consuming, and labor intensive, and generates
significant amounts of chemical waste. These drawbacks can severely
limit the number of optimizations that can be tried, so the final
drug resulting from the process may not be the best drug that is
possible.
[0008] There is therefore a need across many different disciplines
for a technology to rapidly, effectively, and economically test the
health effects of chemicals. In particular, there is a need for a
technology to test chemicals, especially pharmaceuticals, on human
metabolic enzymes. Furthermore, there is a need to optimize new
drug candidates rapidly and economically.
SUMMARY OF THE INVENTION
[0009] Disclosed herein is a microarray chip that allows rapid,
effective, and economical testing of the biological effects of
chemicals, including pharmaceuticals. The invention can also be
used to rapidly and economically synthesize variations of drug
candidates and test their biological effects.
[0010] An apparatus of the invention includes one or more test
compositions, and a plurality of independent, permeable
micromatrices, that each encapsulate at least one test
composition.
[0011] A method of the invention is high-throughput screening using
the disclosed apparatus to detect a reaction having a desired
feature. The method includes combining one or more distinct applied
compositions with the micromatrices of the apparatus under
conditions suitable for reacting the applied compositions with the
test compositions. Another step is assaying each reaction above for
a desired feature.
[0012] The advantages of the invention disclosed herein are
significant. The invention combines rapid testing of chemicals for
pharmaceutical benefit, toxicity, side effects, and interactions
between drugs. By providing microarrays, the invention allows the
use of microscopically small amounts of expensive enzymes and
chemicals. By encapsulating test compositions in micromatrices, the
invention allows precious constituents to be reused. By combining
cell-based assays with a microarray, the invention allows
biologically relevant results to be obtained directly.
[0013] Furthermore, the invention provides for a significant and
surprising advance in high-throughput lead optimization of drug
candidates. The invention allows multiple drug candidates to be
chemically modified, producing a range of drug variants, which can
then be directly screened for improved pharmaceutical benefits and
reduced side effects.
DETAILED DESCRIPTION OF THE INVENTION
[0014] A description of preferred embodiments of the invention
follows.
[0015] The invention generally is related to a method and system
for conducting high-throughput, microscale chemical reactions, and
detecting a desired feature of each reaction. The invention can be
used, for example, to test side effects of a drug in humans. A
reaction between a drug and an encapsulated human metabolic enzyme
on the apparatus can produce a product, called a metabolite. If a
cell-based assay using human cells is applied to the apparatus, and
the cells at a location are killed or otherwise undergo a
measurable physiological or morphological change by the metabolite
produced at that location, it indicates that the drug will likely
have an effect, which may be toxicity. The invention can also be
used, for example, to optimize a potential drug candidate or
pharmacophore to improve its efficacy and/or reduce its side
effects. For example, a promising anticancer drug can be applied to
the apparatus. A reaction between the drug and each encapsulated
enzyme in the array can produce an array of closely related drugs.
A cell based assay including, for example, cancerous cells, can be
applied to the array. In this case, death of the cells at a
particular location indicates likely anticancer activity of the
compound produced by the initial drug and the enzyme at that
location. An apparatus can be constructed that combines this
approach with the side effect test, thereby producing a new, more
effective drug and simultaneously testing the new drug for side
effects.
[0016] As used herein, an independent micromatrix is a piece of
matrix material that is less than about one microliter in volume. A
micromatrix is generally greater than 1 picoliter in volume, and
generally not less than about 100 picoliters. Alternatively, a
micromatrix is less than about 1 microliter, preferably less than
about 500 nanoliters, or less than about 250 nanoliters or less
than about 50 nanoliters or less than about 5 nanoliters in volume.
Alternatively, a micromatrix is less than about 500 picoliters in
volume. Preferably, a micromatrix is between about 250 picoliters
and about 10 nanoliters in volume.
[0017] The material of a micromatrix is permeable to small
molecules, including constituents of applied compositions such as
drugs and their reaction products with test compositions.
Preferably, the micromatrix is impermeable (or substantially
impermeable) to the encapsulated enzyme or other test composition,
thereby retaining, or substantially retaining the test composition
or enzyme from leaching out of the micromatrix. Suitable
micromatrix materials include substituted and unsubstituted solgels
and hydrogels. Each micromatrix can be the same or different
material. The matrix material can be substituted or unsubstituted
and includes a solgel, a hydrogel, a polyacrylamide, a
polyacrylate, a polyvinyl alcohol, polyvinylene, or a polyvinyl
silicate, such as a polyacrylate substituted with a sugar
comprising sucrose, glucose, galactose, trehalose, mannose, or
lactose. In another embodiment, the matrix material is a
substituted or unsubstituted solgel. In a preferred embodiment, the
matrix material is a substituted or unsubstituted solgel containing
an enzymatic activity enhancing amount amount of polyvinyl
alcohol.
[0018] A solgel, for example, is a tetramethoxyorthosilicate, a
methyltrimethoxyorthosilicate, a tetraalkoxyorthosilicate, or a
trialkoxyorthosilicate. A hydrogel is, for example, a
polyacrylamide, a polyacrylate, a sugar-substituted polyacrylate,
or a polyvinyl alcohol. A polysaccharide gel is, for example, an
alginate, a dextran, a starch, a cellulose, a carrageenan, a
poly(hyaluronic acid), a heparin, a guar, or an inulin. Other
polymers include a polyvinylene, a poly(vinyl acetate), a
poly(ethyl vinyl ether, a polyacrylate such as a polymethyl
methacrylate, a polystyrene, a polyvinyl silicate, a polyurethane,
a polyalkanoate, a poly(lactic acid), a poly(3-hydroxybutyrate), or
substituted variations thereof.
[0019] Encapsulation means the test composition is contained
essentially within the volume of a micromatrix. This is an
important distinction from surface immobilization for two reasons.
Encapsulation within the volume of a matrix often maintains the
activity of enzymes better than surface immobilization.
Furthermore, the volume of a matrix can contain far more enzyme
than can be attached to a surface area equal to the footprint of a
micromatrix. More enzyme leads to faster, more complete reactions,
which means, for example, that more reaction products can be
produced, which leads to easier detection. Depending on the matrix
material precursor, a test composition can be physically trapped or
caged, and/or can be covalently attached by a chemical bond, or
tethered. Preferably, a test composition is only physically
trapped, because covalent modification of test compositions, for
example, enzymes, can reduce their activity.
[0020] Appropriate matrix materials, and encapsulation of
compositions therein are described in the literature, including:
U.S. Pat. No. 5,854,030; U.S. Pat. No. 5,618,933; U.S. Pat. No.
5,474,915; Park, C.; Clark, D. 2002 Biotechnol Bioeng., 78,
229-235; Kim, Y; Park, C.; Clark, D. 2001 Biotechnol Bioeng., 73,
331-337; Wang, P., Sergeeva, M. V., Lim, L., and Dordick, J. S.
1997, Nature: Biotechnology 15: 789-793; Novick, S. J. and Dordick,
J. S. 2000, Biotechnol. Bioeng. 68: 665-671; Sergeeva, M. V.,
Paradkar, V. M., and Dordick, J. S. 1997, Enzyme Microb. Technol.
20: 623-628; Novick, S. J. and Dordick, J. S. 1998, Chem. Mat. 10:
955-958; Kim, J., Dedeo, R. and Dordick, J. S. 2002; Biotechnol.
Progress. The entire teaching of the preceding works are
incorporated herein by reference. See Examples 1 and 2 for more
details.
[0021] In another embodiment of the apparatus, the micromatrices
are fixed on a solid support. A solid support can be, for example,
a semiconductor wafer, a glass or quartz microscope slide, a metal
surface, a polymeric surface, a monolayer coating on a surface, the
exterior surface of a probe, the interior surface of a channel or
conduit, and the like. Preferably, the solid support is a flat,
thin solid, such as a glass microscope slide or a silicon wafer.
The micromatrices are also separated on the solid support.
Preferably, the micromatrices are fixed in a regularly spaced,
two-dimensional array on the solid support, for example, located at
the vertices of an imaginary square grid on the surface of the
support.
[0022] Preferably, the solid support includes a physical barrier
that isolates at least one micromatrix from at least one other
micromatrix. For example, each micromatrix, or a group of
micromatrices, can be fixed in a well, channel, conduit, or
depression; or be fixed on a raised platform; or be surrounded
partially or totally by a raised wall or barrier; or surrounded
partially or totally by a depressed channel; or some combination
thereof.
[0023] The inclusion of a physical barrier overcomes a potential
problem with microarrays, namely, controlling "cross-talk" from
mixing or dilution of applied compositions and reaction products
between adjacent micromatrices. This problem can also be overcome
by controlling the volume of liquid used in an applied composition.
For example, if each micromatrix, occupying a volume of about one
microliter, is placed in a microwell of total volume of about ten
microliters, the volume of applied composition should be less than
about nine microliters.
[0024] Alternatively, "cross-talk" can be desirable in a particular
experiment. For example, a drug can be tested for side effects
caused by reaction of its metabolite from one enzyme with a second
enzyme. Two micromatrices can each be located in the same
microwell, or alternatively, in a support without physical
barriers, an excess volume of applied composition can be used so
that the metabolite is washed from its originating micromatrix to
an adjacent micromatrix.
[0025] The distance separating the micromatrices depends on a
number of factors, including the size of the micromatrices, the
resolution of the micromatrix fabrication technique, the volume of
liquid in an applied composition, the presence of physical barriers
in the solid support separating micromatrices, etc. For example, if
the micromatrices are deposited on the solid support by hand, the
spacing will limited by the dexterity of the experimenter. There
are commercially available robotic microarray spotters that can
deliver volumes as small as 100 picoliters or smaller. As a
practical limit, adjacent micromatrices should be separated from
each other by greater than about twice the diameter of the average
spot.
[0026] In another embodiment, two or more micromatrices each
encapsulate a distinct test composition. In an alternative
embodiment, groups of micromatrices are included wherein each
micromatrix within a group encapsulates a distinct test composition
compared to each other micromatrix in its group. For example, a
microarray to determine the response of common metabolic profiles
to a xenobiotic could include 5.times.5 sub-arrays of 25
micromatrices each. Each micromatrix could contain one of 25
enzymes of interest. Each sub-array could differ by the amounts of
each of the 25 enzymes in each micromatrix, or by the specific
amino acid sequence of each of the 25 enzymes, etc. For example,
for a high-throughput capability microarray designed to model 100
different metabolic liver enzyme profiles, 100 sub-arrays per slide
can be prepared, each sub-array representing the liver P450
metabolic profile of an individual, a related group of individuals,
a population subgroup, a pathological profile, and the like. In a
preferred embodiment, each micromatrix encapsulates a distinct
composition.
[0027] Individual micromatrices can be prepared from solutions of
precursors using manual pipetting, but the creation of a microarray
for high throughput analyses can best be accomplished by using a
commercially available robotic microarray spotter. The spotting and
reactions should be performed in a constant humidity chamber within
the robotic spotter, thereby preventing dessication of the solgel
micromatrices once formed. If necessary, glycerol can be added to
the spotting solution to retard evaporation, in an amount between
about 0.01% and about 5% by weight of the total solution. In an
alternative embodiment, the step of encapsulating is selected from
the group consisting of producing a micromatrix in the presence of
one or more distinct test compositions. Another embodiment of the
method is the step of combining one or more distinct test
compositions with a micromatrix material. See Examples 1 and 2 for
more details.
[0028] A robotic microarray spotter can be used in a number of ways
relevant to the invention, including to prepare arrays of
micromatrices on a surface, to add applied compositions to
individual test compositions, to remove multiple samples in
parallel from multiple interaction sites, and to add cell based
assay preparations to the microarray. Of the many commercial
spotters available, there are, for example, contact pin spotters
such as the GeneTAC G.sup.3 (Genomic Solutions, Lansing, Mich.) and
piezoelectric (inkjet mechanism) spotters such as the NANO-PLOTTER
NP1.2.TM. (GeSiM mbH, Grosserkmansdorf, Germany).
[0029] As used herein, test compositions or applied compositions
can be the same or different. Those that are distinct are those
that vary in some measurable physical, chemical, or biological
property and can differ in number of components, molecular formula,
isotopic composition, structural formula, pH, sequence (of amino
acids, DNA bases, RNA bases, monomers, etc), protein folding
structure, presence or absence of cofactors, species, isoform,
lifecycle, tissue origin, cancerous/noncancerous state, and the
like.
[0030] A test composition comprises an indicator, a chemical
compound, a biochemical compound, a catalyst, a cell extract, a
cell fragment, or a cell, where at least one test composition
comprises a constituent of biological origin. A constituent that is
of, for example, biological origin, can be directly derived from an
organism or it can be a chemically synthesized or genetically
engineered copy or analog of a constituent derived form an
organism. Optionally, a test composition comprises a constituent of
mammalian origin. In another alternative, a test composition
comprises a constituent of human origin. In still another
embodiment, a test composition comprise an enzyme, a cofactor, an
antibody, a cell, a cell fragment, or a cell extract.
Alternatively, each test composition comprises at least one enzyme
and its associated cofactor. Preferred enzymes can be anyone one of
those belonging to the six classes of enzymes and include
oxidoreductases, transferases, hydrolases, lyases, isomerases and
ligases. More preferably, each test composition comprises at least
one cytochrome P450 enzyme isoform and its associated cofactor.
Most preferably, the test composition is a single cytochrome P450
enzyme isoform and its associated cofactor.
[0031] An applied composition includes one or more constituents
that have the potential for reaction with a test composition. For
example, if a test composition is a human metabolic enzyme, an
applied composition could be a drug. In general, applied
compositions will contain at least one constituent that may be
termed a xenobiotic, which is any compound that is foreign to an
organism. As used herein, a xenobiotic also includes compounds that
are foreign to an organism's normal function. Examples include a
naturally occurring peptide that has an elevated concentration in a
diseased organism, or a natural protein that has an unnatural
folding configuration, as in a prion-related disease. In one
embodiment, the applied composition further comprises a hydrogel, a
protein gel, a polysaccharide gel, a cellulose, a gelatin, a
polystyrene, or a polyacrylamide. In a preferred embodiment, the
applied composition further comprises a hydrogel selected from the
group consisting of polyvinyl alcohol, collagen, carrageenan,
poly(hyaluronic acid), and inulin. In a preferred embodiment, the
applied composition includes collagen.
[0032] The applied composition can be added to the microarray in a
number of ways. If a single applied composition is to be added to
an array containing distinct test compositions, biocatalytic
reactions can be initiated by dipping the array into a solution
(aqueous, organic, or mixed aqueous-organic cosolvent) of applied
composition and allowing the substrate to diffuse into the printed
biocatalyst. After removing the microarray from the bulk substrate
solution and shaking off excess substrate and/or drying the slide,
bio-transformations of the lead compound proceed within each
matrix. In a second method, the lead compound can be added to the
array using a robotic microarray spotter, which can deliver precise
volumes of a distinct applied composition to a distinct test
composition in the array.
[0033] The volume of applied composition solution added should be
optimized to provide efficient wetting of each micromatrix and
enable effective partitioning of the applied composition into the
micromatrix. For example, the volume of the applied composition
solution added can be between about 0.2 and about 5 times the
volume of each micromatrix. Alternatively, the volume of the
applied composition solution added can be between about 0.5 and
about 2 times the volume of each micromatrix. In a preferred
implementation, the volume of the applied composition solution
should be about the volume of each micromatrix.
[0034] The applied composition solution should be spotted
containing a specific concentration of the active constituent(s).
For example, in a lead optimization, the objective is to chemically
modify a lead compound by catalysis in each micromatrix by each
test composition. Thus, it can be effective to use an applied
composition where the concentration of the active constituent(s)
effectively saturates one or more enzymes in the array. By
contrast, in a toxicity experiment, using a concentration high
enough to saturate the enzymes in the array may not provide
biologically relevant information, particularly if the
concentration is high enough to force saturation binding of even
weakly bound constituents, or if the concentration is much higher
than could conceivably be expected in a biological system. In each
case, the determination of concentration has to be made with
respect to the objectives and the composition of the applied
composition and test compositions.
[0035] A further embodiment of the method comprises the step of
combining a distinct applied composition with a distinct test
composition. Many possible variations are inherent in this
embodiment. For example, an applied composition can be tested
against numerous distinct test compositions in parallel, e.g., a
chip with several thousand distinct encapsulated enzymes could be
dipped in a solution containing a single drug. In another
variation, numerous distinct applied compositions can each be
tested against individual test compositions in parallel. For
example, on a chip with a 10.times.10 array of 10 distinct enzymes,
each enzyme in a column is identical, and a robotic microarray
spotter applies 10 distinct drug candidates across the 10 rows,
leading to 100 distinct reactions. Alternatively, numerous distinct
applied compositions can be combined in a predetermined manner with
numerous distinct test compositions in parallel. For example, a
10.times.10 array can contain 100 distinct enzymes and a robotic
microarray spotter can apply 100 distinct drug candidates, leading
to 100 distinct reactions. Alternatively, the product of a reaction
can be combined with a second test composition, leading to a second
product. For example, after reaction, an aspiration probe can
remove a sample containing a reaction product from a first
encapsulated enzyme, and apply it to a second encapsulated enzyme,
thereby producing a second product.
[0036] Another embodiment of a method of the invention includes the
steps of removing a sample from an interaction site, and applying
the sample to a micromatrix encapsulating a second distinct test
composition. This could be accomplished, for example, by using a
probe, such as an aspiration probe or a contact probe to remove the
sample, and then using the probe to apply the sample to a different
test composition. The probe could be, for example, a single probe
or could be part of an array of probes as part of a robotic
microarray spotter. Alternatively, multiple interactions could be
conducted by using a large excess of applied composition, whereby
the excess solvent of the applied composition directs a product of
the interaction to an adjacent micromatrix.
[0037] In another embodiment, the applied composition further
includes a competitive inhibitor of a constituent of the test
composition. In another embodiment, the test composition includes a
competitive inhibitor of a constituent of the test composition. In
yet another embodiment, the applied composition is combined with a
distinct test composition. In still another embodiment, a distinct
applied composition is combined with the test composition. In a
preferred embodiment, a distinct applied composition is combined
with a distinct test composition.
[0038] Reactions that can be conducted on the disclosed apparatus
include chemical reactions that transform an applied composition
into a reaction product, for example, reacting a drug with a human
metabolic enzyme to produce a drug metabolite. A chemical reaction
also includes temporary interactions between compounds that lead to
a signaling event, such as reversible binding of a substrate by an
enzyme that leads to a color change. To conduct a reaction, an
applied composition, for example, a drug, is applied to each
micromatrix encapsulated enzyme in an array.
[0039] The method disclosed herein can be used to conduct numerous
specific kinds of chemical reactions include, among others,
condensation, acylation, dimerization, alkylation, rearrangement,
transposition, decarbonylation, coupling, aromatization,
epoxidation, disproportionation, hydrogenation, oxidation,
reduction, substitution, isomerization, stereoisomerization,
functional group conversion, functional group addition,
elimination, bond cleavage, photolysis, photodimerization,
cyclization, hydrolysis, polymerization, binding, such as between a
receptor and a ligand; inhibition, such as between an enzyme and an
inhibitor; recognition, such as between an antibody and a hapten;
activation, such as between an agonist and a receptor;
inactivation, such as between an antagonist and a receptor; and the
like.
[0040] Conditions suitable for conducting reactions include
physical conditions such as temperature, pressure, and reaction
time. Also included are chemical conditions such as concentration,
solvents, and consumable reagents such as a co-substrate, enzyme
cofactors, pH, consumable reagents (such as adenosine triphosphate
and nicotinamide adenine dinucleotide phosphate), and the like. In
the context of cell-based assays, suitable conditions include
temperature, water, growth time, growth nutrients, and the
like.
[0041] An embodiment of the disclosed apparatus includes a
detector. A detector assays a desired feature, i.e., physical,
chemical, or biological evidence of reactions, for example, color
changes due to binding of a drug by an antibody, molecular weights
of drug metabolites produced by a metabolic enzyme, or the toxicity
of a drug metabolite to cancerous cells. A detector comprises an
aspiration probe, a laser desorption probe, an ion beam desorption
probe, a gas desorption probe, a liquid desorption probe, a contact
probe, an optical spectrometer, a microscope, an imager, a mass
spectrometer, a chromatography apparatus, an electrochemical
detector, a particle detector, a chemical affinity detector, a
radiation detector, a magnetic resonance spectrometer, a cell
proliferation assay, a cytotoxicity assay, an immunoassay, a
binding assay, or a staining assay. Some of the components
comprised by the detector, such as the various probes, are not
necessarily detectors per se but function to remove a sample and
direct it to another component of the detector. In an alternative
embodiment, the detector comprises an aspiration probe, an optical
spectrometer, a microscope, an imager, a mass spectrometer, or a
cell based assay, such as a cell proliferation assay or a
cytotoxicity assay. In a preferred embodiment, the detector
includes a cell proliferation assay or a cytotoxicity assay.
[0042] In the disclosed method, desired features of reactions can
be assayed via detection in situ or by removing a sample for
analysis. In situ detection can be conducted by the detector of the
disclosed apparatus. For detection off the apparatus, samples can
be removed using aspiration, laser desorption, ion beam desorption,
gas desorption, liquid desorption, contact removal, and the like.
Aspiration, for example, removes a liquid sample by drawing a
vacuum, laser desorption uses laser energy to volatilize a sample
from a solid or liquid phase into a gas phase, contact removal
applies a probe to a sample, whereupon a portion of the sample
adheres to the probe, gas desorption, similar to aspiration,
directs a gas across a site to entrain a sample in the gas stream,
and the like. Removed samples can be assayed by the detector of the
disclosed apparatus, or by another detector. A particular
embodiment involves the use of a cell based assay, which includes
detecting cell proliferation, cell death (cytotoxicity), and other
metabolic or morphological changes in cells. These assays can be
performed using both cell monolayer overlays, which cover at least
a portion of the apparatus, and gel droplets, which cover only one
micromatrix. Cells used in the monolayer overlays and the gel
droplets can be cultured in natural or synthetic gels including a
hydrogel, a protein gel, a polysaccharide gel, a cellulose, a
gelatin, a polystyrene, or a polyacrylamide.
[0043] Thus, the invention also includes the embodiment where the
apparatus or microarray are overlayed or covered by a second matrix
containing a second test composition. Suitable test compositions
include those employed in the micromatrices. The second matrix
applied to the microarray as a single layer or film across the
entire substrate. Alternatively, the second matrix can be added in
discrete and independent droplets over each micromatrix, thereby
permitting the test compositions in the second matrix to be
different.
[0044] The P450 isoforms found in the human liver provides a
representative example of how test composition constituents can be
selected and used in the present invention. The human liver
includes 16 major isoforms responsible for the vast majority of
xenobiotic metabolism (Table 1). A summary of the relative amounts
of P450 isoforms responsible for drug metabolism in the uninduced
human liver is given in Table 2. This distribution can be
reproduced in micromatrix sub-arrays. Further, this capability can
be expanded to accommodate differences in P450 isoform levels, and
mutations among isoforms, allowing investigation of the influence
of P450 variability on drug metabolism in an individual, a related
group of individuals, a population subgroup, a pathological
profile, and the like.
TABLE-US-00001 TABLE 1 Summary of Commerically Available P450
Isoforms, their Substrates (Xenobiotics), and Known Inhibitors P450
Representative Substrates Isoform (fluorogenic ones given in bold)
Representative Inhibitors 1A1 PAHS (e.g., benzo[a]pyrene, pyrene),
7- Ellipticine ethoxyresofuffin 1A2 Aromatic amines, PAHs,
caffeine, Furafylline, verapamil, diltiazern coumadin, 3-cyano-7
etboxycoumarin 2A6 Coumarin, nicotine, steriods, valproic acid
Trancypromine, diethyldithiocarbarnate 2C8 Paclitaxel, ibuprofen,
dibenzylflourescein Quercitin, omeprazole 2C9 Dieolfenac,
ibuprofen, omeprazolc, Sulfaphenaole, cimetidine, coumadin,
tamoxifen, dibenzylfluorescein fluotetine, valproic acid 2C18
Imipramine, naproxen, omeprazole Cimetidine, fluoxetine, omeprazole
2D6 Capropril, dextramethorphan, tramadol, Qunidine, codeine,
codein, 3-[2-(n.sub.3N-diethyl-N- haloperidol, valproic acid
methylamine)ethyl]-7-methoxy-4- methylcoumarin 2E1 Acetaminophen,
chlorzoxezone, 7- Diethylidithiocarbamate,
methoxy-4-trifuloromethylcoumarin ritonavir 3A4 Atorvastain,
cortisol, cyclophosphamide, Ketoconzaole, digitoxin, indinavir,
loratidine, lovastatin, erythromycin, fluconazole paclitaxel,
tamosifen, testoterone, terfenadine, dibenzylfluorescein 3A5
Cortisol, lovastatin, terfenadine Ketoconazole, Miconazole 3A7
Cortisol, lovastatin, terfenadine Ketoconazole, miconazole 4A11
Lauric acid 1-Aminobenzotriazole 4F2 Arachadonic acid, Leukotriene
B.sub.4 17-Octadecynoic acid 4F3A & B Leukotriene B.sub.4
Quercitin, ketoconzaole
TABLE-US-00002 TABLE 2 Representative Distribution of P450 Isoforms
in the Human Liver.sup.34 Average % of P450 Isoform Total Liver
P450 1A2 13 2A6 4 2B6 1 2C8, 2C9, 2C18, 2C19 18 2D6 2.5 2E1 7 3A4,
3A5 28
[0045] In the foregoing, each test composition contains an
individual P450 isoform. To more accurately represent targets of
interest, for example, the in vivo environment of the liver,
different and/or multiple combinations of P450 isoforms can be
included in each distinct test composition. For example, a
5.times.5 sub-array format can be used to examine metabolites of a
drug or xenobiotic in the presence of different levels and ratios
of P450 isoforms. For example, an applied composition can contain
cyclophosphamide (a prodrug precursor to 4-hydroxycyclophosphamide)
in combination with a 5.times.5 sub-array wherein each of the 25
micromatrices encapsulate different levels of CYP3A4 and CYP2B6,
two human liver P450 isoforms. The 5.times.5 sub-array can be
prepared where each micromatrix in the cluster contains either or
both of the two P450 isoforms, and the relative amounts of the two
P450 isoforms can be adjusted by spotting different ratios. Upon
adding cyclophosphamide, this can result in a secondary reaction by
the second P450 isoform or result in inhibition of the second P450
isoform. In another alternative, if only one isoform is used per
test composition, an excess of the applied composition solution can
be added, whereby products from reaction with one isoform, by means
of the excess volume, contact adjacent test compositions. Either
alternative can approximate the behavior of cyclophosphamide
metabolism in the human liver. See Example 2 for more details.
[0046] In the foregoing, liver P450 enzymes were used as particular
illustrative examples, due to their importance in human metabolism.
However, this should not be construed as a limitation. For example,
a wide variety of other enzymes from other organs, and other
organisms can be used, as cited above. Enzymes that recognize
substrates instead of transforming them, such as receptors, can be
used. Catalysts other than protein enzymes can be used, such as
catalytic antibodies, chemical catalysts, or RNA enzymes. Cell
extracts that contain multiple cell components can be encapsulated,
providing for multi-step interactions of applied compositions.
[0047] The application of cell based assays, including cytotoxicity
and cell proliferation, as detection techniques in the disclosed
method can be understood in the following implementation of a cell
proliferation assay. The cells to be used in the assay can be
entrapped in hydrogel droplets, which can be spotted directly over
each interaction site. The use of hydrogels to support and restrain
the cells allows cell based assays to be applied to each
micromatrix site, whereby each assay responds to the products
associated with the micromatrix it covers and therefore each assay
result can be distinguished. A hydrogel is a matrix material, such
as collagen, hyaluronic acid, polyvinyl alcohol, polysaccharides,
etc, that be used to support and restrain cells in a specific area.
Note that the hydrogel matrix material used in a cell-based assay,
while potentially made of the same material as the micromatrices,
is distinct from the micromatrices.
[0048] Following application of the hydrogel droplet culture, the
cells can be allowed to grow for an extended period of time, e.g.,
one week, while exchanging the growth medium according to standard
protocols, during which time growth can be monitored by standard
staining and image analysis techniques. The determination of an
appropriate incubation time is an individual experimental decision
based on standard protocols for the cells in use and the
experiment's objectives.
[0049] There are a wide variety of cells that can be used in such
assays. Determination of which cell to use depends on the purpose
of the particular experiment. For example, in optimizing a new
cancer drug lead, one experiment would use a cytotoxicity assay
employing cancerous cells, where cell death is the sought after
result. In another experiment, the same array can be used in
combination with normal cells, for example, for the same organ as
the cancerous cells, in order to determine the toxicity of the
optimized drug leads; here, cell proliferation is the desired
result. Correlation of the two experiments allows optimized lead
compounds to be ranked according to their desirable toxicity to
cancer cells vs. undesirable toxicity to normal cells. Cells that
can be used, or the tissues/organs they can be derived from,
include, but are not limited to bone marrow, skin, cartilage,
tendon, bone, muscle (including cardiac muscle), blood vessels,
corneal, neural, brain, gastrointestinal, renal, liver, pancreatic
(including islet cells), cardiac, lung, pituitary, thyroid,
adrenal, lymphatic, salivary, ovarian, testicular, cervical,
bladder, endometrial, prostate, vulval, esophageal, etc. Also
included are the various cells of the immune system, such as T
lymphocytes, B lymphocytes, polymorphonuclear leukocytes,
macrophages, and dendritic cells. In addition to human cells, or
other mammalian cells, other organisms can be used. For example, in
optimizing a pesticide lead compound, nerve cells from the target
organism could be used. In another example, in testing for
environmental effects of an industrial chemical, aquatic
microorganisms that could be exposed to the chemical can be used.
In still another example, organisms such as bacteria that are
genetically engineered to possess or lack a certain trait could be
used. For example, in the optimization of an antibacterial lead
compound for combating antibiotic resistant organisms, the cell
assay could include cells that have been engineered to express one
or more genes for antibacterial resistance.
[0050] In an anti-cancer drug lead optimization, for example, a
cytotoxicity assay will use cancerous cells. Examples of cells that
could be used include a breast cancer cell line (MCF7), a human
hepatocyte (HepG2 cells), and a kidney cell line (A-498 cells).
MCF7 cells can be grown as monolayers in T-25 flasks containing
buffered phenol-red free DMEM medium supplemented with 10% (v/v)
fetal bovine serum, glucose (4.5 g/L), and glutamine (2 mM) in a
humidified incubator at 5% (v/v) CO.sub.2 at 37.degree. C. The cell
culture medium should not be supplemented with antibiotics.
Monolayer cultures of HepG2, a human hepatoblastoma cell line, can
be grown in DMEM medium as recommended by the American Type Culture
Collection and described by Scharnagl et al. (2001).sup.32, except
that antibiotics should not be added to the medium. ATCC recommends
against using antibiotics when culturing HepG2 cells (ATCC
Technical Services), and, in general, antibiotics can undergo
undesirable biotransformations catalyzed by enzymes in the
micromatrices. A-498 cells, a human kidney carcinoma cell line, can
be grown in supplemented DMEM as recommended by ATCC.
[0051] After allowing reactions between applied compositions and
test compositions to proceed, cell monolayers can be transferred
from the tissue culture flask to the microarray for cytotoxicity
assays. The transfer procedure is be similar to that described by
Ziauddin and Sabatini (2001).sup.33 for cell microarrays expressing
defined cDNAs. The microarray can then be immersed in a tissue
culture dish containing the appropriate medium, incubated at
37.degree. C. for 48 h, and stained for viability.
[0052] A potential problem with the hydrogel cell assay methods can
be cross talk, similar to that discussed in a preceding section on
the solid support. Here, mixing and dilution of test
composition-generated products can occur in the liquid medium
surrounding the individual drops. Three scenarios could be
possible: (a) the collagen gel is surrounded by excess liquid
medium (i.e., the chip can be immersed in a solution of medium
during the growth-inhibition assays); (b) the growth medium is
confined to the collagen gel only, thus preventing possible
transport of compounds from one collagen-gel droplet to another; or
(c) as disclosed for the apparatus, each site or subarray of sites
on the microarray is provided with a physical barrier, such as a
microwell, whereby the accompanying liquid growth medium is
contained by the barrier.
[0053] In scenario (a), there is no cross talk because the
particular interaction to be studied involves applied compositions
or subsequent products that diffuse slowly on the timescale of the
analysis. Scenario (c) was discussed in a preceding section.
Scenario (b) requires that cells are able to grow in a
medium-filled hydrogel matrix without the presence of a surrounding
liquid reservoir. For example, hydrogel drops can be prepared and
inoculated with cells as described previously, with the following
modifications. A cell suspension (containing ca. 4.times.10.sup.5
cells/mL) can be combined with UV-sterilized collagen solution and
30.times. medium (30.times. DMEM with 10% FBS). Collagen gel drops
containing different concentrations of medium can be prepared by
mixing these reagents in the following proportions: 0.1 mL cell
suspension+1 mL collagen+0.2 mL 30.times. medium; 0.1 mL cell
suspension+0.8 mL collagen+0.4 mL 30.times. medium; 0.1 mL cell
suspension+0.6 mL collagen+0.6 mL 30.times. medium. The collagen
spotted slides can then be incubated in 5% (v/v) CO.sub.2 at
37.degree. C. for up to 3 days, and the cells stained for viability
with a Live/Dead test kit (Molecular Probes). See Example 2 for
more details.
Exemplification
[0054] The present invention is illustrated by the following
examples, which are not intended to be limiting in any way.
Example 1
Solgel Enzyme Microarrays
[0055] Solgel micromatrices containing active enzymes were
stabilized on glass at near-neutral pH and room temperature. A
multi-well bilayer of polydimethylsiloxane (PDMS) was used to
support the matrix array and contain the reaction medium. The
enzymes in the solgels were catalytically representative of their
solution counterparts; a good linear correlation (R=0.98) was
obtained when the activity of the solgel enzymes were plotted
against the activity of the soluble hydrolases. The solgel arrays
were reusable and exhibited greater thermostability when compared
to soluble enzymes. The enzyme-containing solgel arrays were
further miniaturized by spotting micromatrices on microscope
slides. An enzyme-containing solgel microarray was generated
containing 300 solgel micromatrices on a glass microscope
slide.
Example 2
P450 Microarrays
[0056] Sol solution was prepared by mixing 25 .mu.l
methyltrimethoxysilane (MTMOS) with 10 .mu.l polyvinyl alcohol
(PVA, MW 10,000) in distilled water (10% w/w). The resulting sol
had a pH of 2, and the formed gel was then neutralized quickly by
washing with aqueous buffer. To prevent detachment of solgel
matrices from the glass slide and to make hemispherical matrices,
MTMOS solution (pH7) was spin coated (2 .mu.l at 3000 rpm for 30 s)
onto the glass. The reactions were performed in arrays containing
150 solgel matrices, each with a volume of 1 .mu.l prepared using a
manual micropipetter. P450 activity was tested as follows: 0.5
.mu.l green fluorescent substrate (2 mM, DBOMF, a fluorescein
analog), 2.5 .mu.l NADP.sup.+ (10 mM) and 2.5 .mu.l regeneration
system (glucose 6-phosphate dehydrogenase plus glucose 6-phosphate)
were added to 94.5 .mu.l phosphate buffer (200 mM, pH 8). P450
activity was assayed by spotting 5 .mu.l applied composition
solution onto the 1 .mu.l solgel matrix containing the P450 (0.14
pmol or ca. 5.6 .mu.g/mL of the hydroxlase component), and the
relative fluorescence intensity was monitored vs. time using a
plate reader (the glass slide sitting atop a 384-well plate) at an
excitation wavelength of 485 nm and emission wavelength of 535
mn.
[0057] The reactivity of CYP3A4 in the solgel matrix compared to
the enzyme in aqueous solution is summarized in Table 3. The
intrinsic activity of the P450 in the solgel was high, with a
V.sub.max nearly identical to the native enzyme formulation in
aqueous solution. Thus, the process of incorporating the
multicomponent CYP3A4 test composition into the solgel did not
affect the V.sub.max of the enzyme. Moreover, the enzyme reaction
was not limited by diffusion; calculation of the Observable Modulus
yielded a value less than 1, indicating that the reaction was
kinetically limited.
TABLE-US-00003 TABLE 3 Kinetic Constants of CYP3A4 Enzyme V.sub.max
K.sub.m V.sub.max/K.sub.m Form (nmol/min/nmol P450) (.mu.M)
(min.sup.-1) Soluble 0.69 12.7 0.060 Solgel 0.61 215 0.0031
[0058] In addition to the kinetic constants summarized in Table 3,
the following data has been obtained for sol-gel preparations of 1
.mu.l and below. These values are given in Table 4. These results
were obtained by spotting sols onto a glass microscope slide using
a microarray spotter.
TABLE-US-00004 TABLE 4 Additional data Initial Rate Solgel Volume
(nM/min/nM-P450 1000 nL 0.85 125 nL 0.80 100 nL 0.75 25 nL 0.70
Example 3
Cell Growth in Collagen-Gel Droplets and Pro-Drug Activation
[0059] Solgel matrices were prepared as described above, except for
one modification involving spin coating. Specifically, the MTMOS
spin coat can be hydrophobic and can be insufficiently wetted by
the collagen gel, thereby resulting in poor attachment of the
collagen gel onto the slide. To prevent detachment of solgel
matrices from the glass slide and to produce hemispherical
matrices, polymaleic anhydride-alt-.alpha.-olefin (PMA-OL) in
toluene was spin coated (2 ml at 3000 rpm for 30 s) onto the glass.
The P450 reactions (involving CYP3A4) were then performed in arrays
containing 40 solgel matrices, each with a volume of 1 .mu.l
prepared using a manual micropipetter. For the P450 reaction, 5
.mu.l substrate solution (1 mM cyclophosphamide and 2 mM NADPH) was
spotted on the P450 solgel and incubated for 2 h at 30.degree. C.
to produce 4-hydroxycyclophosphamide as a product toxic to MCF7
breast cancer cells.sup.31. Cyclophosphamide is a known prodrug
against MCF7 cells and can be metabolized to active compounds, such
as 4-hydroxycyclophosphamide, by CYP3A4 in the liver (Scheme
I).
##STR00001##
[0060] Scheme 1 shows the CYP3A4-catalyzed metabolism of
cyclophosphamide. The primary metabolite is
4-hydroxycyclophosphamide, which is in equilibrium with
aldophosphamide. The aldophosphamide spontaneously decomposes into
phosporamide mustard, the alkylating agent, and acrolein.
[0061] Preparation and overlay of the collagen-gel matrices
containing MCF7 cells was carried out by trypsinizing a confluent
layer of MCF7 cells from a T-25 cell-culture flask, centrifuging
the cell solution for 10 min at 800 rpm, and re-suspending the
cells in 1 mL of FBS-supplemented DMEM medium. The cell suspension
(0.2 mL, containing ca. 4.times.10.sup.5 cells/mL) was then
combined with 2 mL of UV-sterilized collagen solution (from rat
tail) and 0.4 mL of 10.times. medium (10.times. DMEM with 10% FBS)
adjusted to pH 7. Collagen gel droplets (5 .mu.L, containing ca.
900 cells) were then spotted on top of each solgel matrix. After 30
min pre-incubation at room temperature; the spotted slide was
overlaid with growth medium and incubated for two days.
[0062] After two days the medium was discarded and the Live/Dead
test kit (Molecular
[0063] Probes) was used to produce a green fluorescent response by
living cells and a red fluorescent signal by dead cells. To this
end, 20 .mu.l of ethidium homodimer-1 (2 mM) and 5 .mu.l of calcein
AM (4 mM) were added to 10 mL of sterile tissue-culture grade PBS
buffer, and 5 .mu.L of this mixture was applied to each collagen
drop. Following incubation at 37.degree. for 30 min, each collagen
gel matrix was observed with fluorescence microscopy. There was a
significant increase in the number of dead cells (red spots) and
the ratio of dead to live cells in the collagen gel matrix that
contains CYP3A4.
[0064] These results indicate that CYP3A4 can be sufficiently
active in solgel matrices to transform cyclophosphamide into its
4-hydroxy derivative, which is toxic to MCF7 breast cancer
cells.
Example 4
Design of a Factorial Experiment to Optimize a Particular Array
[0065] Enzymes encapsulated in solgels can be active and stable
with a V.sub.max nearly as high as in aqueous solution. To optimize
enzyme activity and extend these preliminary results to other
enzyme isoforms, a broad factorial design can be desirable to
elucidate the effects of solgel formulation conditions on test
composition activity and stability. For example, for p450 isoforms,
key variables, and the range of parameters to be studied are
summarized in Table 5. A second-order factorial design was used to
study the influence of factors that have been identified as being
critical in influencing P450 enzyme activity and stability:
H.sub.2O/MTMOS ratio, MTMPS/TMOS ratio, solution pH poly(vinyl
alcohol) (PVA) concentration, and P450 concentration. Using
commercially available fluorogenic enzyme variants can facilitate
this optimization phase.
[0066] In an example of a factorial design, two levels and five
factors yield 2.sup.5 experiments to be performed. In this
optimization stage, the solgels can be arrayed manually to give 150
micromatrices per microscope slide using a 384-well plate as a
visual template for the micromatrices. This enables use a
fluorescent plate reader for the P450 assays. Kinetic constants
(V.sub.max and K.sub.m) and the observed half-life at room
temperature of CYP3A4 can then be determined for each of the 32
experimental conditions to be studied. The moderate-throughput
manual spotting can be suitable for this number of experiments of
CYP3A4, as well as other P450 isoforms (see Table 1).
TABLE-US-00005 TABLE 5 Factors and Factor Settings for a 2.sup.5
Factorial Design Factor Low setting High setting H.sub.2O/MTMOS
ratio (v/v) 0.5 3 MTMOS/TMOS ratio (v/v) 0.25 3 Solution pH 2 8 PVA
concentration (w/v) 1 10 P450 concentration (nmol/mL) 0.01 0.2
[0067] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *