U.S. patent application number 09/951131 was filed with the patent office on 2003-04-10 for microcantilever apparatus and methods for detection of enzymes.
This patent application is currently assigned to Protiveris, Inc.. Invention is credited to Bottomley, Lawrence A., Ghosh, Madhushree, Saul, Richard, Shen, Shanxiang.
Application Number | 20030068655 09/951131 |
Document ID | / |
Family ID | 25491300 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030068655 |
Kind Code |
A1 |
Bottomley, Lawrence A. ; et
al. |
April 10, 2003 |
Microcantilever apparatus and methods for detection of enzymes
Abstract
An apparatus and a method are provided for detecting an enzyme
by measuring a change in defection of a microcantilever having a
substrate for the enzyme.
Inventors: |
Bottomley, Lawrence A.;
(Lawrenceville, GA) ; Ghosh, Madhushree;
(Rockville, MD) ; Shen, Shanxiang; (Bethesda,
MD) ; Saul, Richard; (Gaithersburg, MD) |
Correspondence
Address: |
Sonia K Guterman Esq
Mintz, Levin, Cohn, Ferris, Glovsky and Popeo P C
One Financial Center
Boston
MA
02111
US
|
Assignee: |
Protiveris, Inc.
|
Family ID: |
25491300 |
Appl. No.: |
09/951131 |
Filed: |
September 12, 2001 |
Current U.S.
Class: |
435/7.9 ;
427/2.11 |
Current CPC
Class: |
G01N 33/54373 20130101;
C12Q 1/00 20130101 |
Class at
Publication: |
435/7.9 ;
427/2.11 |
International
Class: |
G01N 033/53; G01N
033/542; C12Q 001/70; B05D 003/00 |
Claims
What is claimed is:
1. A Method for detecting an enzyme, the method comprising:
depositing a coating material on a first surface of at least one
microcantilever; adding at least one substrate to the coating
material, the substrate capable of interacting with the enzyme;
exposing the microcantilever with the substrate to a sample; and
measuring a deflection of the microcantilever, wherein the
deflection indicates the presence of the enzyme in the sample.
2. A method according to claim 1, wherein adding the substrate
comprises adding at least one biomaterial.
3. A method according to claim 2, wherein adding the biomaterial is
adding a substance selected from the group consisting of a nucleic
acid, a protein, a lipid, a hydrocarbon, and a polysaccharide.
4. A method according to claim 1, wherein adding the substrate is
adding a drug.
5. A method according to claim 1, wherein the deflection is caused
by a change in stress on the surface of the microcantilever.
6. A method according to claim 5, wherein measuring the deflection
is observing the change by a means selected from the group
consisting of an optical means, an electron tunneling means, a
capacitive means, a piezoelectric means, and a piezoresistive
means.
7. A method according to claim 6, wherein measuring the deflection
is using the optical means.
8. A method according to claim 7, wherein the optical means
comprises a laser.
9. A method according to claim 1, further comprising analyzing the
deflection of the microcantilever as a function of a time parameter
determined from the time of exposing the microcantilever to the
sample.
10. A method according to claim 9, wherein analyzing the deflection
comprises using a microprocessor adapted for comparing,
calculating, and storing the deflection of the microcantilever as a
function of a time parameter.
11. A method according to claim 9, wherein analyzing the deflection
further comprises analyzing a parameter selected from the group of:
concentration of enzyme, concentration of substrate, presence of a
cofactor and presence of an inhibitor.
12. A method according to claim 1, wherein the at least one
microcantilever has a length of about 1 .mu.m to about 500 .mu.m, a
width of about 1 .mu.m to about 50 .mu.m, and a thickness of about
0.1 .mu.m to about 10 .mu.m.
13. A method according to claim 1, wherein depositing the coating
material further comprises depositing a metal.
14. A method according to claim 13, wherein the metal is selected
from at least one of the group consisting of aluminum, copper,
gold, chromium, titanium, silver, and mercury.
15. A method according to claim 14, wherein the metal is gold.
16. A method according to claim 1, wherein depositing the coating
material further comprises depositing a plurality of metals.
17. A method according to claim 16, wherein depositing a plurality
of metals further comprises depositing a first layer of chromium
and a second layer of gold.
18. A method according to claim 16, wherein depositing a plurality
of metals further comprises depositing a first layer of titanium
and a second layer of gold.
19. A method according to claim 13, wherein the metal is an amalgam
or an alloy.
20. A method according to claim 1, wherein the microcantilever has
a second surface selected from the group consisting of aluminum:
oxide, iridium oxide, silicon, silicon oxide, silicon nitride,
tantalum pentoxide, and a plastic polymer.
21. A method according to claim 1, further comprising prior to
adding the substrate to the first surface, reacting the
microcantilever with a bifunctional cross-linker, the bifunctional
cross-linker capable of further reacting with the substrate.
22. A method according to claim 21, wherein the bifunctional
cross-linker is selected from the group consisting of:
dithiobis(succinimido undecanoate (DSU); long chain
succinimido-6-[3-(2-pyridyldithio)-propiona- mido] hexanoate
(LCSPDP); succinimidyl-6-[3-(2-pyridyldithio)propionamido]
hexanoate (SPDP); and m-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS).
23. A method according to claim 21, wherein the bifunctional
cross-linker is DSU.
24. A method according to claim 1, wherein adding the substrate is
designing the microcantilever for detecting an enzyme selected from
the group consisting of: a hydrolase, an oxidoreductase, a
transferase, a lyase, and a ligase.
25. A method according to claim 24, wherein the enzyme is a
hydrolase.
26. A method according to claim 25, wherein the hydrolase is a
protease.
27. A method according to claim 1, wherein the enzyme is selected
from the group of consisting of: a kinase, a phosphatase, an
endopeptidase, an exopeptidase, a restriction endonuclease, an
exonuclease, and a polymerase.
28. A method according to claim 26, wherein the protease is a
metalloprotease or a serine protease.
29. A method according to claim 24, wherein the transferase is
selected from the group consisting of: a glycosyl transferase, a
glutathione S-transferase, an acetyl transferase, and a DNA methyl
transferase.
30. A method according to claim 24, wherein the lyase is selected
from the group consisting of: a polysaccharide lyase, a
3-hydroxy-3-methylglutaryl CoA lyase, an argininosuccinate lyase
and an isocitrate lyase.
31. A method according to claim 24, wherein the oxidoreductase is
selected from the group consisting of: a hydroxylamine
oxidoreductase, a glyphosphate oxidoreductase, a quinine
oxidoreductase, an ubiquinone oxidoreductase, and a protein
disulfide oxidoreductase.
32. A method according to claim 1, wherein the sample comprises an
enzyme that is substantially purified.
33. A method according to claim 1, wherein the sample comprises a
biological fluid.
34. A method according to claim 33, wherein the biological fluid is
selected from the group consisting of: a cell lysate, a culture
medium, a spent medium, an animal extract, and a plant extract.
35. A method according to claim 33, wherein the biological fluid
comprises a bodily fluid from a vertebrate animal.
36. A method according to claim 35, wherein the vertebrate is a
mammal.
37. A method according to claim 36, wherein the mammal is a
human.
38. A method according to claim 35, wherein the bodily fluid is
selected from the group consisting of: blood, lymph, tissue fluid,
urine, bile, sweat, synovial fluid, amniotic fluid, abdominal
fluid, pericardial fluid, pleural fluid, cerebrospinal fluid,
gastric juice, intestinal juice, joint cavity fluid, tears, and
nasal discharge.
39. A method according to claim 1, wherein the enzyme is associated
with a medical condition in a vertebrate animal.
40. A method according to claim 39, wherein the medical condition
is a genetic defect.
41. A method according to claim 40, wherein the medical condition
is selected from the group consisting of: cystic fibrosis, Fabry
disease, Gaucher disease, Tay-Sachs disease, sickle cell anemia,
Lesch-Nyhan disease, Parkinsons disease, amyotrophic lateral
sclerosis, Crohns disease, diabetes mellitus, mannosidosis disease,
celiac disease, X-linked glomerular disease, and
mucopolysaccharidosis.
42. A method according claim 39, wherein the medical condition is a
cancer.
43. A method according to claim 42, wherein the cancer is selected
from the group consisting of a cancer of the: brain, liver,
pancreas, lung, prostate, and breast.
44. A method according to claim 43, wherein the cancer is prostate
cancer and the enzyme is prostate specific antigen.
45. A method according to claim 43, wherein the cancer is breast
cancer, and the enzyme is a collagenase.
46. A method according to claim 39, wherein the medical condition
is the presence of an infectious agent.
47. A method according to claim 46, wherein the infectious agent is
selected from the group consisting of: a virus, a bacterium, a
fungus, a protozoan, and a helminth.
48. A method for detecting in a sample an associating substance
that binds to a substrate, wherein detecting the substance involves
at least one microcantilever configured to be responsive to a
micro-force, the method comprising: depositing a coating material
on a first surface of the microcantilever; adding at least one
substrate to the coating material, the substrate capable of
interaction with the substance; exposing the microcantilever with
the substrate to the sample; and measuring a resulting free surface
energy change on the surface of the microcantilever, wherein the
surface energy change indicates binding to the substrate by the
associating substance in the sample.
49. A method according to claim 48, wherein the associating
substance is selected from the group consisting of: a binding
protein, an enzyme, a cofactor, a receptor ligand, an antibody, a
polysaccharide, a lipid, a nucleic acid, and a steroid.
50. A method according to claim 48, wherein the associating
substance is an enzyme.
51. A method according to claim 50, wherein the enzyme binds the
substrate and fails to dissociate.
52. A method according to claim 51, wherein the enzyme has no
activity on the substrate.
53. A method according to claim 48, wherein the substrate is a
non-cleavable pseudosubstrate.
54. A method according to claim 1, wherein the at least one
microcantilever is a block array having a plurality of
microcantilevers.
55. A method according to claim 48, wherein the substrate is a
plurality of biomaterials.
56. A method according to claim 55, wherein the substrate comprises
an inhibitor of enzymatic activity.
57. A method of screening for an inhibitor of an enzyme having a
substrate on a microcantilever having a coating, the method
comprising: adding the substrate to a first side of a first and a
second microcantilever, the substrate capable of interacting with
the enzyme; exposing the first microcantilever with the substrate
to a sample, the sample containing a candidate inhibitor and the
enzyme; and measuring a deflection of the first microcantilever in
comparison to a deflection of a second microcantilever exposed to
the enzyme in the absence of the candidate inhibitor.
58. A method according to claim 57, wherein the first
microcantilever and the second microcantilever are located in a
first and second interaction well of a microfluidics device.
59. A method according to claim 58, wherein a third microcantilever
and a fourth microcantilever are located in a third and fourth
interaction well, the third and fourth wells having a different
concentration of enzyme than the first and second wells.
60. A method according to claim 58, wherein a third microcantilever
and a fourth microcantilever are located in a third and fourth
interaction well, the third and fourth wells having different
samples comprising candidate inhibitors.
61. An apparatus to measure a microforce generated by an
interaction between an enzyme and a biomaterial, comprising: at
least one microcantilever, wherein said microcantilever has a
length, a width, and a thickness; a coating material deposited on a
first surface of the microcantilever; a biomaterial capable of
attachment to the coating material; and at least one interaction
well, wherein the microcantilever with coating material and the
biomaterial is exposed to a sample, the sample comprising the
enzyme.
62. An apparatus according to claim 61, wherein the biomaterial
comprises an enzymatic substrate.
63. An apparatus according to claim 61, wherein the biomaterial
comprises an enzymatic pseudosubstrate.
64. An apparatus according to claim 61, wherein the at least one
microcantilever further comprises of a block array having a
plurality of microcantilevers.
65. An apparatus according to claim 61, wherein the microcantilever
length is about 1 .mu.m to about 500 .mu.m, the width is about 1
.mu.m to about 50 .mu.m, and the thickness is about 0.1 .mu.m to
about 10 .mu.m.
66. An apparatus according to claim 64, wherein the coating
material is selected from at least one of the group consisting of
copper, gold, aluminum, chromium, titanium, silver, and
mercury.
67. An apparatus according to claim 66, wherein the coating
material is gold.
68. An apparatus according to claim 61, wherein a second surface of
the microcantilever is selected from the group consisting of
silicon, silicon nitride, other silicon compounds, metal compounds,
gallium arsenide, germanium, germanium dioxide, glass, zinc oxide,
diamond, quartz, paladium and a plastic polymer.
69. An apparatus according to claim 61, wherein the apparatus is
disposable.
70. An apparatus according to claim 61, wherein the apparatus is
reusable.
Description
TECHNICAL FIELD
[0001] The general field of the invention relates to an apparatus
and method for detecting the presence of an enzyme in a sample by
measuring a deflection of a microcantilever, the surface of the
microcantilever having a substrate for the enzyme. In various
embodiments, the invention is of use in proteomics, drug discovery,
medical research, medical, veterinary, and dental diagnostics,
forensics, and military applications.
BACKGROUND
[0002] A large variety of enzymes are important in medicine,
industry, and other applications. Discovery of novel enzymes has
gone hand-in-hand with development of certain industries, for
example as the discovery of bacterial restriction enzymes and the
development of genetic engineering. Enzymes are important in
various medical pathologies (Fang J., et al., Proc. Natl. Acad.
Sci. U.S. 97: 3884-3889, 2000), as novel therapeutics (U.S. Pat.
No. 6,210,667 issued Apr. 3, 2001), as targets for development of
novel therapeutic agents, such as HIV protease (U.S. Pat. No.
6,271,235 issued Aug. 7, 2001), in industrial processes such as
antibiotic biosynthesis (U.S. Pat. No. 6,258,555 issued Jul. 10,
2001), degradation of unwanted materials such as polyurethane (U.S.
Pat. No. 6,180,381, issued Jan. 30, 2001) and in the food industry
(U.S. Pat. No. 5,827,712, issued Oct. 27, 1998). The need to obtain
novel enzyme activities is so great that protein engineering
research has been directed toward development of catalytic
antibodies (U.S. Pat. No. 5,807,688, issued Sep. 15, 1998).
[0003] Thin bimorph microcantilevers can undergo bending
(deflection) due to differential stresses following exposure to and
binding of a compound from their environment, for example in a
fluid sample. Soft microcantilevers having spring constants less
than 0.1 N/m are sensitive to stress differentials that arise as a
result of interactions between extremely small amounts of a
substrate material on a surface of the microcantilever and one or
more materials in sample. For a given microcantilever with a
specially designed coating layer, the deflection yields information
about components of the environment to which the microcantilever is
exposed. Microcantilevers are capable of detecting calorimetric
enzyme-mediated catalytic biological reactions with femtoJoule
resolution. (Thundat et al., "Microcantilever Sensors", Microscale
Thermophysical Engr. 1, pgs. 185-199, 1997.) Oligonucleotide
interactions within a sample can be detected using a monolithic
array of test sites formed on a surface to which the sample is
applied (U.S. Pat. No. 5,653,939).
[0004] There is a need for methods and an apparatus for detecting
an interaction between an enzyme and its enzymatic substrate, or
detecting a protein having an enzymatic activity or a related
molecule, such as a catalytic antibody, or a binding protein, as
measured by a response of a microcantilever to the stress change
caused changes in free surface energy and bonding energy. There is
a need in medical and veterinary diagnostics, and in research, for
detection and analysis of binding and activities of enzymes and
enzyme-like proteins.
SUMMARY
[0005] The invention in one embodiment provides a method for
detecting an enzyme, the method comprising: depositing a coating
material on a first surface of at least one microcantilever; adding
at least one substrate to the coating material, the substrate
capable of interacting with the enzyme; exposing the
microcantilever with the substrate to a sample; and measuring a
deflection of the microcantilever, wherein the deflection indicates
the presence of the enzyme in the sample. In a related embodiment,
adding the substrate comprises adding at least one biomaterial, a
biomaterial selected from the group consisting of a nucleic acid, a
protein, a lipid, a hydrocarbon, and a polysaccharide, for example.
In another related embodiment, the substrate is a drug.
[0006] In a related embodiment of this method, the deflection is
caused by a change in stress on the surface of the microcantilever.
In a preferred embodiment, the deflection is measured by observing
the change by an optical means, which preferably includes a laser.
Alternatively, an electron tunneling means, a capacitive means, a
piezoelectric means or a piezoresistive means may be used to
observe the change in deflection.
[0007] In a related embodiment, the method further comprises
analyzing the deflection of the microcantilever as a function of a
time parameter determined from the time of exposing the
microcantilever to the sample. Analyzing the deflection comprises
using a microprocessor adapted for comparing, calculating, and
storing the deflection of the microcantilever as a function of a
time parameter. Analyzing the deflection further comprises
analyzing a parameter selected from the group of: concentration of
enzyme, concentration of substrate, presence of a cofactor and
presence of an inhibitor.
[0008] In the related embodiment, the method comprises the at least
one microcantilever having a length of about 1 .mu.m to about 500
.mu.m, a width of about 1 .mu.m to about 50 .mu.m, and a thickness
of about 0.1 .mu.m to about 10 .mu.m. Depositing the coating
material further comprises depositing a metal. The metal is
selected from at least one of the group consisting of aluminum,
copper, gold, chromium, titanium, silver, and mercury. For example,
the metal is gold.
[0009] In a related embodiment, the method further comprises
depositing a plurality of metals. Depositing a plurality of metals
further comprises depositing a first layer of chromium and a second
layer of gold. In a related embodiment, the method further
comprises depositing a first layer of titanium and a second layer
of gold. The metal in other embodiments is an amalgam or an
alloy.
[0010] In a related embodiment, the microcantilever has a second
surface selected from the group consisting of aluminum: oxide,
iridium oxide, silicon, silicon oxide, silicon nitride, tantalum
pentoxide, and a plastic polymer.
[0011] In a related embodiment, the method further comprises, prior
to adding the substrate to the first surface, reacting the
microcantilever with a bifunctional cross-linker, the bifunctional
cross-linker capable of further reacting with the substrate. The
bifunctional cross-linker is selected from the group consisting of:
dithiobis(succinimido undecanoate (DSU); long chain
succinimido-6-[3-(2-pyridyldithio)-propionamido] hexanoate
(LCSPDP); succinimidyl-6-[3-(2pyridyldithio)-propionamido]
hexanoate (SPDP); and m-maleimidobenzoyl-N-hydroxysuccinimide
ester. For example, the bifunctional cross-linker is DSU.
[0012] In a related embodiment of the method, the microcantilever
detects an enzyme selected from the group consisting of: a
hydrolase, an oxidoreductase, a transferase, a lyase, and a ligase.
For example, the enzyme is a hydrolase. The hydrolase is a
protease. For example, the protease is a metalloprotease or a
serine protease. Further, the enzyme is selected from the group of
consisting of: a kinase, a phosphatase, an endopeptidase, an
exopeptidase, a restriction endonuclease, an exonuclease, and a
polymerase.
[0013] The transferase is selected from the group consisting of: a
glycosyl transferase, a glutathione S-transferase, an acetyl
transferase, and a DNA methyl transferase. For example, the lyase
is selected from the group consisting of: a polysaccharide lyase, a
3-hydroxy-3-methylglutaryl CoA lyase, an argininosuccinate lyase
and an isocitrate lyase. For example, the oxidoreductase is
selected from the group consisting of: a hydroxylamine
oxidoreductase, a glyphosphate oxidoreductase, a quinine
oxidoreductase, a ubiquinone oxidoreductase, and a protein
disulfide oxidoreductase. In a related embodiment of the invention
of the invention, the sample comprises an enzyme that is
substantially purified. According to a further embodiment of the
method, the sample comprises a biological fluid. The biological
fluid is selected from the group consisting of: a cell lysate, a
culture medium, a spent medium, an animal extract, and a plant
extract. For example, the biological fluid comprises a bodily fluid
from a vertebrate animal, such as a human or other mammal.
According to an embodiment provided by this method, the bodily
fluid is selected from the group consisting of: blood, lymph,
tissue fluid, urine, bile, sweat, synovial fluid, amniotic fluid,
abdominal fluid, pericardial fluid, pleural fluid, cerebrospinal
fluid, gastric juice, intestinal juice, joint cavity fluid, tears,
and nasal discharge.
[0014] In a related embodiment the invention, the enzyme is
associated with a medical condition in a vertebrate animal. The
medical condition is a genetic defect for example, the medical
condition is selected from the group consisting of: cystic
fibrosis, Fabry disease, Gaucher disease, sickle cell anemia,
Lesch-Nyhan disease, Tay-Sachs disease, Parkinsons disease,
amyotrophic lateral sclerosis, Crohns disease, diabetes mellitus,
mannosidosis disease, celiac disease, X-linked glomerular disease,
and mucopolysaccharidosis. In another embodiment, the medical
condition is a cancer, for example, the cancer is selected from a
cancer of the brain, liver, pancreas, lung, prostate, or breast. In
related embodiment, the cancer is prostate, and the enzyme is
prostate specific antigen. In a related embodiment, the cancer is
breast cancer, and the enzyme is a collagenase. The medical
condition in another embodiment is the presence of an infectious
agent. For example, the infectious agent is selected from the group
consisting of: a virus, a bacterium, a fungus, a protozoan, and a
helminth.
[0015] An embodiment of the invention provides a method for
detecting in a sample an associating substance that binds to a
substrate, wherein detecting the substance involves at least one
microcantilever configured to be responsive to a micro-force, the
method comprising: depositing a coating material on a first surface
of the microcantilever; adding at least one substrate to the
coating material, the substrate capable of interaction with the
substance; exposing the microcantilever with the substrate to the
sample; and measuring a resulting free surface energy change on the
surface of the microcantilever, wherein the surface energy change
indicates binding to the substrate by the associating substance in
the sample.
[0016] In a related embodiment of the invention, the associating
substance is selected from the group consisting of: a binding
protein, an enzyme, a cofactor, a receptor ligand, an antibody, a
polysaccharide, a lipid, a nucleic acid, and a steroid. For
example, the associating substance is an enzyme wherein the enzyme
binds the substrate and fails to dissociate. In another example,
the enzyme has no activity on the substrate. In yet another
example, the substrate is a non-cleavable pseudosubstrate.
[0017] In a related embodiment of, the invention provides at least
one microcantilever which is a block array having a plurality of
microcantilevers. The substrate in a related embodiment is a
plurality of biomaterials. The substrate comprises an inhibitor of
enzymatic activity.
[0018] In one embodiment, the invention provides a method of
screening for an inhibitor of an enzyme, wherein detecting the
inhibitor involves having a substrate for the enzyme on a
microcantilever, the method comprising: adding the substrate to a
first side of a first microcantilever having a coating, the
substrate capable of interacting with the enzyme and with the
coating; exposing the first microcantilever with the substrate to a
sample, the sample containing a candidate inhibitor and the enzyme;
and measuring a deflection of the first microcantilever in
comparison to a deflection of a second microcantilever exposed to
the enzyme in the absence of the candidate inhibitor. In a related
embodiment of the method, the first microcantilever and the second
microcantilever are located in a first and second interaction well
of a microfluidics device. In a related embodiment, a third
microcantilever and a fourth microcantilever are located in a third
and fourth interaction well, the third and fourth wells having
different concentrations of enzyme than the first and second wells.
In a related embodiment, third microcantilever and a fourth
microcantilever are located in a third and fourth interaction well,
the third and fourth wells having different samples of candidate
inhibitors than the first and second wells.
[0019] An embodiment of the invention provides an apparatus to
measure a microforce generated by an interaction between an enzyme
and a biomaterial, comprising: at least one microcantilever,
wherein said microcantilever has a length, a width, and a
thickness; a coating material deposited on a first surface of the
microcantilever; a biomaterial capable of attachment to the coating
material; and at least one interaction well, wherein the
microcantilever with the coating material and the biomaterial is
exposed to a sample, the sample comprising the enzyme. The
biomaterial comprises an enzymatic substrate. The biomaterial
comprises an enzymatic pseudosubstrate. The at least one
microcantilever comprises of a block array having a plurality of
microcantilevers. The microcantilever length is about 1 .mu.m to
about 500 .mu.m, the width is about 1 .mu.m to about 50 .mu.m, and
the thickness is about 0.1 .mu.m to about 10 .mu.m. The
microcantilever coating is selected from at least one of the group
consisting of copper, gold, aluminum, chromium, titanium, silver,
and mercury. For example, the coating is gold coating. A further
embodiment of this apparatus, a second surface of the
microcantilever is selected from the group consisting of silicon,
silicon nitride, other silicon compounds, metal compounds, gallium
arsenide, germanium, germanium dioxide, glass, zinc oxide, diamond,
quartz, paladium and a plastic polymer. The apparatus in one
embodiment is disposable. In another embodiment, the apparatus is
reusable.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a schematic representation of a partial top view
of a microcantilever showing three dimensions, first and second
surfaces, and substrate molecules deposited on the first
surface.
[0021] FIG. 2 is a schematic diagram of a side view of a
microcantilever having molecules of a bifunctional cross-linking
agent attached to the surface of the microcantilever and to a
biomaterial, and the biomaterial bound directly to the surface of
the microcantilever. Various types of enzymes and modes of binding
to and digesting substrate molecules are shown.
[0022] FIG. 3 is a schematic view of a microcantilever showing
various positions of potential deflection and return to an original
position.
[0023] FIG. 4 is a time course (in seconds, on the abscissa) of
microcantilever deflection (in nm, on the ordinate) as a result of
papain digestion of IgG (upper function), compared to a prior
control using the same microcantilever exposed to buffer only
(lower function).
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0024] FIG. 1 shows a microcantilever having a first surface 101, a
second surface 105, a height 102, a width 104, and a length 109.
The first surface can have at least one coating 106. An enzymatic
substrate 108 is affixed to the first surface directly 107, or by
covalent reaction with a bifunctional cross-linking agent 110.
Non-covalently bound substrate molecules can be washed from the
first surface following a reaction with the cross-linking agent,
for example by use of a buffer having a low pH, or a mild
detergent. Covalent linking of substrate molecules, rather than
direct binding, is a preferred embodiment, as the former process
produces a more geometrically homogeneous array of substrate
molecules.
[0025] FIG. 2 illustrates the interaction of classes of enzymes
with substrate molecules on a first surface of a microcantilever,
following addition of an enzyme sample to the microcantilever.
Prior to addition of sample, all substrate molecules are
full-length, as shown in Panel A, second substrate molecule from
right. Panel A shows an enzyme as a cross-hatched circle, binding
to a recognition site on the interior of the substrate molecule
(substrate molecule at left), and cleaving the substrate molecule,
leaving a shorter product covalently attached to the surface
(second, third, fifth, seventh, etc., substrate molecules from
left). This result would be obtained from digestion of a DNA
substrate molecule by an endonuclease such as a restriction enzyme,
e.g., BamH1 or EcoR1, or from digestion of a protein substrate
molecule by a protease such as trypsin. Panel B shows an enzyme as
a cross-hatched circle, binding to a free end of a substrate
molecule distal from the attached end, and cleaving the substrate
processively. This result would be obtained from digestion of a
protein substrate molecule by, for example, an exonuclease or an
exopeptidase. Panel C shows interaction of binding proteins (open
or stippled circles), or inactive enzymes, with substrate
molecules. Following binding, no digestion of substrate molecules
is obtained.
[0026] FIG. 3 shows deflection of a microcantilever from an initial
position, A. Addition of substrate molecules to a first surface can
alter the position of the microcantilever to a new position, e.g.,
position B or position C. Subsequent enzyme digestion as in FIG. 2,
panel A, or FIG. 2, panel B, can further alter the deflection,
e.g., from position B to position C, or from position C to position
A. Binding of inactive enzyme or of a binding protein to the
substrate can alter the position of the microcantilever, causing
deflection, for example, from position C to position B.
[0027] FIG. 4 shows a time course of deflection of a
microcantilever, to which the first surface of which has been
covalently attached a protein substrate, a solution of IgG antibody
molecules. The microcantilever having covalently attached antibody
is first exposed to a control buffer (lower function), as a result
of which exposure no change is observed in the deflection. The same
microcantilever, then exposed to an appropriate concentration of
the protease papain, as described in Example 1. The data show a
significant change in deflection, of about 60 nm, occurring over a
time course of several minutes following exposure to the
papain.
[0028] Definitions
[0029] Unless the context otherwise requires, as used in this
description and in the following claims, the terms below shall have
the meanings as set forth below.
[0030] The term "microcantilever" is a structural term that refers
to a flexible beam that may be bar-shaped, V-shaped, or have other
shapes, depending on its application. One end of the
microcantilever is fixed on a supporting base, another end standing
freely. Microcantilevers are usually of microscopic dimensions, for
example, they can be about 1 .mu.m to about 50 .mu.m, about 20
.mu.m to about 150 .mu.m, about 50 .mu.m to about 250 .mu.m, about
100 .mu.m to about 400 .mu.m, about 200 .mu.m to about 500 .mu.m,
or about 250 .mu.m to about 750 .mu.m. Further, the width can be,
for example, about 1 .mu.m to about 50 .mu.m, about 5 .mu.m to
about 20 .mu.m, about 10 .mu.m to about 30 .mu.m, about 20 .mu.m to
about 50 .mu.m, or about 25 .mu.m to about 100 .mu.m. Silicon and
silicon nitride are the most common molecules used to fabricate
microcantilevers with an atomically sharp tip mounted near the
freestanding end. Other molecules have also been reported for
making microcantilevers, including piezoelectric molecules, plastic
molecules and various metals.
[0031] Specifically, microcantilevers can be manufactured from a
variety of materials, including for example, from ceramics,
silicon, silicon nitride, other silicon compounds, metal compounds,
gallium arsenide, germanium, germanium dioxide, zinc oxide,
diamond, quartz, palladium, tantalum pentoxide, and plastic
polymers. Plastics can include: polystyrene, polyimide, epoxy,
polynorbornene, polycyclobutene, polymethyl methacrylate,
polycarbonate, polyvinylidene fluoride, polytetrafluoroethylene,
polyphenylene ether, polyethylene terephthalate, polyethylene
naphthalate, polypyrrole, and polythiophene. Microcantilevers can
be obtained that are custom fabricated such as Diffraction Ltd.,
Waitsfield, Vt., for example.
[0032] Microcantilevers with a compound immobilized on the surface
on the free end have been used to detect and screen receptor/ligand
interactions, antibody/antigen interactions and nucleic acid
interactions (U.S. Pat. No. 5,992,226, issued on Nov. 30, 1999).
Deflection is measured using optical and piezoelectric methods.
Microcantilevers can measure concentrations using electrical
methods to detect phase difference signals that can be matched with
natural resonant frequencies (U.S. Pat. No. 6,041,642, issued Mar.
28, 2000.) Determining a concentration of a target species using a
change in resonant properties of a microcantilever on which a known
molecule is disposed, for example, a biomolecule selected from DNA,
RNA, and protein, is described in U.S. Pat. No. 5,763,768.
[0033] A method and apparatus for detecting and measuring physical
and chemical parameters in a sample media uses micromechanical
potentiometric sensors (U.S. Pat. No. 6,016,686, issued Jan. 25,
2000). Chemical detection of a chemical analyte, is described in
U.S. Pat. No. 5,923,421, issued Jul. 13, 1999. Magnetic and
electrical monitoring of radioimmune assays, using antibodies
specific for target species which cause microcantilever deflection,
e.g., magnetic beads binding the target to the microcantilever, are
described in U.S. Pat. No. 5,807,758, issued Sep. 15, 1998.
[0034] The contents of all cited references are hereby incorporated
by reference herein.
[0035] The term "first surface" as used herein refers to that
geometric surface of a microcantilever designed to receive and bind
to molecules of a substrate for an enzyme. One or more coatings can
be deposited upon this first surface. Thus the term "second
surface" refers to the area of the opposite side of the
microcantilever which is designed not to contain coating or enzyme
substrates. As the second surface is generally not coated, it is
generally comprised of the material from which the microcantilever
or microcantilever array is fabricated, prior to any coating
procedure applied to the first surface. Alternatively, it may be
coated with a material different from the first surface's
coating.
[0036] A first surface of a microcantilever can be fabricated to
have an intermediate layer, for example, sandwiched between the
first surface comprising for example, gold, and the second surface,
comprising for example silicon nitride. The intermediate layer may
be an alloy comprising a plurality of metals, for example, the
intermediate layer may be an amalgam comprising mercury with at
least one of, chromium, silver, and titanium.
[0037] U.S. Pat. Nos. 6,096,559 issued Aug. 1, 2000, and 6,050,722
issued Apr. 18, 2000, describe fabrication of a microcantilever,
including use of material such as ceramics, plastic polymers,
quartz, silicon nitride, silicon, silicon oxide, aluminum oxide,
tantalum pentoxide, germanium, germanium dioxide, gallium arsenide,
zinc oxide, and silicon compounds. Coating of micromechanical
sensors with various interactive molecules is described in U.S.
Pat. No. 6,118,124, issued Sep. 12, 2000.
[0038] Deflection or bending of a microcantilever from a first
position to at least a second position may be due to differential
stress on a first surface of the microcantilever in comparison to a
second surface, the change in surface stress resulting from
exposure of the microcantilever to a component of a particular
environment. A microcantilever can be deflected by changing from a
first environment to a second environment. For example, the
environment can be altered in many possible ways including: an
enzyme can be added or deleted or the enzyme concentration can be
lowered or raised; a specific co-factor of an enzyme can be added
or deleted or the concentration of the co-factor can be lowered or
raised; a specific inhibitor of an enzyme can be added or deleted
or the concentration of the inhibitor can be lowered or raised; a
sample can be diluted or concentrated prior to, during or after
exposure to a microcantilever; a sample can experience a
temperature change prior to, during or after exposure to a
microcantilever; a sample can experience a change in pH prior to,
during or after exposure to a microcantilever; a sample can
experience a change in conductivity prior to, during or after
exposure to a microcantilever; and a sample can experience a change
in viscosity prior to, during or after exposure to a
microcantilever.
[0039] Measuring a deflection is measuring the distance moved or
change in position of a microcantilever that alters from a first
occupied position, at which first position the microcantilever with
the biomaterial on the first surface of the microcantilever has not
yet bound or reacted with the enzyme, to a second position occupied
by the microcantilever after it has altered its position because of
binding to or reaction of the biomaterial on the microcantilever
with the enzyme in the environment, and consequent alter action of
the biomaterial.
[0040] A deflection characteristic is a pattern of deflection of a
microcantilever which is reproducible in extent of distance
traveled, for example as measured in nm, and frequency per unit
time. The deflection characteristic can distinguish specific
conditions of enzyme and substrate, and further reaction conditions
such as temperature, concentration, ionic strength, presence of an
ion or other co-factor, preservatives, and other conditions
well-known to one of skill in the enzymological arts. The extent of
a deflection under a particular set of these conditions can become
a signature for a specific reaction. A deflection characteristic is
calculated from a measurement of extent of movement of the
microcantilever, as a function of the time of addition of a sample,
or as an extent of the movement as a function of concentration of
an enzyme, of concentration of a substrate, of concentration of an
inhibitor, of concentration of a co-factor, of pH, or of
temperature, and the like.
[0041] A microprocessor can be included in an apparatus or a
method, such that an integrated circuit containing the arithmetic,
logic, and control circuitry required to interpret and execute
instructions from a computer program. The microprocessor components
of the measuring devices reside in an apparatus for detection of
microcantilever deflection. Detection of an enzyme in an
environment The term "environment" means the entire complex of
factors to which the microcantilever is exposed. For example, the
complex of factors may include a sample having a substance such as:
a substantially purified enzyme; a bodily fluid containing at least
one enzyme; a substantially pure inhibitor; a bodily fluid
containing an inhibitor, and combinations of such components, and
the like. The term "environment" also includes the concentration of
each of any of components of the sample that can affect enzyme
activity. Factors such as temperature of the environment, while
contributing to stress, are controlled by standard means, well
known to one of ordinary skill in the art, such as use of an
insulated and thermally controllable housing, and by monitoring of
deflection of a reference microcantilever in an environment
designed to omit either the substrate, the enzyme, or an essential
co-factor. The reference microcantilever may be exposed to
inactivated enzyme, or it may contain a control enzyme compared to
that found in the sample. The difference between the environments
of the reference microcantilever and the experimental
microcantilever results in a measure of the amount of deflection
experienced by the experimental microcantilevers compared to the
deflection seen in the reference environment as the background for
which all other microcantilever deflections are measured.
[0042] As used herein, deflection of a microcantilever from a first
position to at least a second position can occur by a physical or
chemical alteration of an enzyme substrate molecule linked to a
surface of a microcantilever. For example, a physical alteration
which is a change in mass of the sensor material, e.g., of a
substrate molecule, can be incurred in when a DNA substrate reacts
with either a DNA nuclease, such as an endonuclease or an
exonuclease, or with a DNA ligase. In the first case, the
alteraction is a reduction in the molecular weight of material on
the microcantilever. In the second case, the alteration is increase
in the molecular weight of material on the microcantilever.
Deflection of the microcantilever changes also when a nuclease
enzyme molecule binds to the DNA molecule. Following digestion of
the substrate and release or removal of the enzyme, deflection of
the microcantilever to another position can be observed. Similarly,
deflection of the microcantilever can change from a first position
to a second position due to a change in mechanical stress from
additional weight on the surface when a substrate interacts with
and binds the enzyme. Deflection can change from a second position
to at least a third position, following, for example, activity of a
ligase molecule results in addition of a length of DNA to the DNA
substrate molecule. Deflection can change from a third position to
at least a fourth position when the ligase dissociates from the DNA
substrate.
[0043] Another embodiment of a deflection of a microcantilever is
observed when, for example, a physical alteration of a substrate
molecule is incurred when a DNA substrate reacts with a DNA
endonuclease or exonuclease. Deflection of the microcantilever can
change from a first position to at least a second position due to
the increased weight on the surface when the substrate interacts
with the enzyme. Deflection can change from a second position to at
least a third position when the nuclease removes DNA from the DNA
substrate molecule. Deflection can change from a third position to
at least a fourth position when the nuclease disassociates from the
DNA substrate. However, as these interactions occur at nsec to /sec
speeds, real time monitoring of deflection is a measurement of an
overall change in the substrate due to the enzymatic activity of
the enzyme.
[0044] The deflection of a microcantilever can be measured by a
means that is capacitive, piezoelectric, piezoresistive, or
optical. The term "capacitive" means storage of energy in a
non-conducting material resulting from a force or stress on the
surface of the material. This force or stress can result in a
deflection of the microcantilever. The term "piezoelectric" means a
voltage and/or current produced between surfaces of a solid
non-conducting material when a mechanical stress is applied to it.
The term "piezoresistive" means a change in electrical resistance
of a substance when a pressure or force is exerted on the surface
of the substance. Optical means include use of ambient ligant and
other sources of light, including lasers. Detection of
microcantilever deflection by optical, electrical and mechanical
means is shown in U.S. Pat. No. 5,653,939 issued Aug. 5, 1997. Use
of laser light sources is shown in U.S. Pat. Nos. 6,016,686 issued
Jan. 25, 2000, and 6,123,819, issued Sep. 26, 2000. Majumdar et al.
(WO 01/14823 A1 international publication date Mar. 1, 2001) uses
measurement of defraction of incident light to measure microforces
with a set of microcantilever finger array blocks that can deflect
relative to a set of fixed frame fingers. Magnetic and electrical
means for detection of deflection are shown in U.S. Pat. Nos.
5,807,758 issued Sep. 15, 1998, 5,156,810 issued Oct. 20, 1992, and
in 5,981,297, issued Nov. 9, 1999, and 6,107,000 issued Aug. 22,
2000, respectively. Piezoelectric means for measuring deflection
are shown in U.S. Pat. Nos. 5,814,525 issued Sep. 29, 1998;
5,445,008 issued Aug. 29, 1995; and 5,719,324, issued Feb. 17,
1998, respectively.
[0045] A time parameter is a time interval for measuring an event
or an occurrence from a first point of time to at least a second
point of time, and also third, a fourth, etc., points in time. In
general, the first point in time is noted as the time of exposing
the microcantilever to the sample.
[0046] A stress is a force exerted on a surface of a
microcantilever which can be associated with intermolecular
interactions on that surface, such as: enzymatic alteration a
substrate on a first surface of a microcantilever, followed by
enzyme release; or, irreversible binding of a protein in a sample
to the substrate. Stress includes any type of force exerted on a
surface of a microcantilever resulting from the interaction of a
specific enzyme substrate, or a specific enzyme inhibitor, or a
potential substrate, with an enzyme. Microcantilevers are sensitive
to stress differentials due to different extents of interaction of
a component of a sample, with one or more materials that have been
added to a coating layer on a first material.
[0047] The term "responsive" means that the microcantilever,
including all coatings and sensor materials such as a substrate for
an enzyme, is sensitive to the stress generated by an interaction
force that arises when an enzyme specifically interacts with the
substrate. The resulting force may comprise chemical-mechanical
forces, thermomechanical forces, electrostatic forces, magnetic
forces, and other types of forces, alone or in combination.
[0048] Enzymes
[0049] The term "enzyme" encompasses a large number of protein
biological catalysts, which are known to or are predicted to
catalyze a reaction. Most commonly, an enzyme can catalyze at least
one of many different possible biochemical reactions that comprise
biological pathways. Further, an enzyme can catalyze an organic
chemical reaction, such as conversion of ethanol to acetic acid, or
an inorganic reaction, such as reduction of molecular nitrogen.
[0050] The molecules that are the results of an enzymatically
catalyzed reaction are referred to as "products." The terms enzyme,
substrate, and product are standard terms in the arts of enzymology
and biochemistry. The term enzyme can include, for example, an
active enzyme in a sample capable of modifying its enzymatic
substrate to yield an enzymatic product on a microcantilever; a
genetically altered enzyme having a catalytic defect; an enzyme
lacking a cofactor essential for catalytic activity; and an enzyme
in a sample binding irreversibly to a pseudosubstrate. The
interaction forces generated by enzyme activity on a substrate
molecule may comprise chemical-mechanical forces,
thermal-mechanical forces, electrostatic forces, magnetic forces,
and other types of forces.
[0051] Enzymes encompass six general classes based on the reaction
being catalyzed, including: isomerases, oxidoreductases,
transferases, hydrolases, lyases, and ligases. Isomerases catalyze
the conversion of a substrate which is a chemical compound, to a
different chemical compound product that contains the same number
and type of atoms, but in a different structural configuration.
Oxidoreductases are involved in oxidation, reduction, and electron
or proton transfer reactions of the substrate. Transferases
catalyze reactions in which groups of atoms are transferred to or
from substrate molecules. Hydrolases cleave one or more of a
variety of covalent bonds of the substrate by hydrolysis. Ligases
join two or more substrate components to form a covalent bond, each
component being part of a substrate complex. Enzymes that are known
in the art can be purified from cells that have been collected and
concentrated as the enzymes are thus purified. Cells are ruptured
by methods commonly employed by artisans in microbiology and cell
biology, for example, sonication, French press, freeze thawing, and
detergent lysis.
[0052] Secreted microbial enzymes can be obtained from spent
culture medium, i.e., growth medium from which cells have been
removed following culture and growth of cells. Enzymes can be
purified by procedures including column chromatography,
particularly affinity column chromatography, and also ion-exchange
column chromatography, size exclusion column chromatography, and,
as fusion proteins using highly specific affinity ligands (see New
England Biolabs Catalog, 2000-2001, pp. 142-143).
[0053] Enzymes are purified and stored in suitable buffers
containing anti-oxidant agents, such as dithiothreitol or
mercaptoethanol, to maintain native cysteine disulfide bonds in a
reduces condition, and with chelators such as EDTA to protect the
enzyme from heavy metal inactivation. Enzymes can be stored at
-20.degree. C. or -70.degree. C., with an agent such as glycerol or
DMSO to prevent water crystal formation, or in a suitable buffer.
Many enzymes of interest are commercially available (Sigma Aldrich,
Inc., St. Louis, Mo.; Calbiochem, San Diego, Calif.; New England
Biolabs, Inc. Beverly, Mass.), as are suitable buffers for storage
and concentrated reaction mixes that are formulated for optimal
enzyme activity and include appropriate ions. Alternatively,
enzymes are available as purified crystals, which can be dissolved
in a suitable buffer at a specific appropriate concentration prior
to use.
[0054] Enzymes herein include in scope any genetically engineered
or semi-synthetic peptide-containing molecule capable of reacting
with another molecule to promote a chemical change, for example, a
catalytic antibody. The term enzyme is further envisioned to
include an activity which has not yet been characterized, but for
which a substrate and assay system can be devised, for example, a
DNA restriction endonuclease that recognizes and binds to a
palindromic or non-palindromic sequence consisting of 10 or more
nucleotides. Further, the term enzyme includes naturally-occuring
or genetically engineered derivatives of an enzyme with known
activity, including a derivative however having reduced or
essentially no activity.
[0055] Enzymes having a known activity are characterized using the
methods and apparatuses herein by parameters of that activity
associated with a particular enzymatic substrate, including
affinity for the substrate, and rate of turnover of the substrate
to yield product. The parameters are known as K.sub.m (Michaelis
constant) as a measure of affinity for a substrate and V.sub.max,
which is a maximum velocity. These parameters are determined by
analyses of enzyme activity as a function of concentrations of
enzyme and substrate, and by observing the reaction as a function
of time. Mutated enzymes, and active enzymes in the presence of an
enzyme inhibitor, can exhibit a lower affinity for a particular
substrate (increased K.sub.m) or a lower turnover number (decreased
V.sub.max). The methods and apparatus of the present invention can
be optimized to determine changes in K.sub.m and V.sub.max of
enzyme derivatives, and for identification and analysis of enzyme
inhibitors.
[0056] Substrates for Enzymatic Activity
[0057] The term "substrate" means a molecule specifically chosen by
one of ordinary skill in the biochemistry of enzymes, because it is
known to be a substance that reacts with an enzyme of interest. The
molecule of substrate, or mixture of different molecules of
different substrates, can be chosen because at least one of the
types of molecules is known to bind specifically to the active site
of the enzyme, such that the enzyme acts to catalyze a chemical
reaction that alters the substrate. For example, the substrate can
be a particular protein for an enzyme which is protease; or, the
substrate can be a DNA molecule having a particular nucleotide
sequence that can be recognized by molecule for a restriction
endonuclease.
[0058] A substrate can be designed to detect a novel enzymatic
activity, i.e., an enzymatic activity that might be present but is
not currently known to be present in one of a plurality of natural
product samples, or from a library of mutated enzymes. The term
"substrate" is commonly used in the engineering arts as a surface
which acts as a support for another material, for example, in U.S.
Pat. No. 6,123,819, issued Sep. 26, 2000. In the present
application the term "substrate" is used to refer only to a member
of that particular class of molecule which specifically can
interact with an enzyme of choice, and which can be bound by the
enzyme and further chemically altered by a reaction catalyzed by
the enzyme, to yield a product that is chemically different from
the initial substrate material.
[0059] Substrates need not be the natural substrate of an enzyme,
and can be designed according to the particular purpose of the
user, including diagnostics, inhibitor search, purity monitoring,
or novel enzyme discovery. Substrates can be nature-identified,
e.g., a protein in a native configuration, or can be denatured and
further chemically modified. The substrate can also be further
modified for use in other means of detection, for example, a
substrate can be colorigenic, fluorogenic, or radioactive, although
these modifications need not affect an aspect of microcantilever
deflection.
[0060] Under some circumstances it is desirable to have a dense
array of substrate molecules, for example, short substrate
molecules, as opposed to a less dense array of longer substrate
molecules. The kinetics of enzyme digestion of a substrate on a
surface of a microcantilever can depend on the size of the
particular enzyme, for example, the Stokes radius of the enzyme, so
that an optimal extent of density and size of substrate molecules
should be determined by the user experimentally. The density of the
substrate on the first surface of the microcantilever can be
adjusted by varying one or more of the factors, including the
concentration of the enzyme, the temperature of the reaction of
enzyme with cross-linking agent, or the duration of time of this
reaction. Further, the substrate can be a mixture of suitable
molecules, as could be determined by one of skill in the art of
enzymology.
[0061] A sensor material can be deposited on the surface of a
microcantilever, and can interact with a component of a sample, for
example, the sensor material is a biomaterial. In another
embodiment, the sensor material can be any substance with which a
protein, particularly an enzyme can interact, and which can be
immobilized on microcantilever.
[0062] The term "biomaterial" means any organic material isolated
from a natural source, or produced synthetically, or produced
semi-synthetically by chemical synthesis with an organic starting
material. For example, a biomaterial can be isolated from a natural
source such as an animal tissue, a plant, or from bacterial cells,
using technology well known to one skilled in the art. A
biomaterial such as a protein can be synthesized semi-synthetically
using recombinant DNA technology, or in a eukaryotic cell-free
system, methods which are well known to one skilled in the art. A
protein can also be synthesized de novo using solid state or
solution peptides synthesis chemistry, with commercially available
devices and substrates well known to one skilled in the art of
peptide synthesis. A biomaterial can be all or a portion of a cell.
A sensor for the detection of bound E. coli cells immobilized using
antibodies on microfabricated structures is disclosed in Ilic et
al. "Mechanical resonant immunospecific biological detector", Appl.
Phys. Lett. Vol. 77, No. 3, pgs. 450-452, Jul. 17, 2000.
Biomaterials and other sensor materials can be obtained
commercially, or can be produced by the artisan in the
laboratory.
[0063] The phrase "non-cleavable pseudosubstrate" means a molecule
that is chemically similar to a natural substrate of the enzyme,
which can bind the enzyme, but which pseudosubstrate is not altered
chemically. A pseudosubstrate can bind covalently or non-covalently
to the enzyme active site, but cannot be converted to the end
product of the chemical reaction. For example, a proteinaceous
protease inhibitor can act as a pseudosubstrates for a protease,
for example, a synthetic inhibitor can act as a pseudosubstrate for
a cAMP-dependent protein kinase.
[0064] The phrase "substantially pure" means that the enzyme of
interest has been physically manipulated to increase the final
concentration in comparison to the initial concentration, with
respect to other non-enzyme materials, for example, that the enzyme
solution is at least 80% pure, is at least 90% pure, is at least
95% pure, or is at least 99% pure with respect to non-enzyme
components of the solution.
[0065] Samples
[0066] The term "sample" means the components dissolved or
dispersed in a fluid state. A sample of interest can be assayed for
the presence of a diagnostically important enzyme in a sample from
a subject; alternatively, a sample can be assayed for presence of a
novel enzyme activity.
[0067] The term "bodily fluid" means any fluid produced or secreted
within or by a body of an animal, blood, lymph, tissue fluid,
urine, bile, sweat, synovial fluid, amniotic fluid, abdominal
fluid, pericardial fluid, pleural fluid, cerebrospinal fluid,
gastric juice, intestinal juice, joint cavity fluid, tears, and
nasal discharge.
[0068] The phrase "medical condition" means any condition in which
the health of a subject is impaired. The medical condition can
include for example a genetic defect, an infection, a cancer which
can be a leukemia or a tumor, and the like.
[0069] The term "infection" is meant to include disorders of a
human or animal subject caused by one or more species of bacteria,
viruses, fungi, or protozoans, which are disease-producing
organisms collectively referred to as "pathogens.".The term "fungi"
is meant to include the yeasts. In this invention, pathogens are
exemplified by, but not limited to: Gram-positive bacteria such as
Enterococcus fecalis, Hemophilus pneumoniae, Listeria
monocytogenes, Mycobacterium tuberculosis, M. leprae,
Proprionibacterium acnes, Staphylococcus aureus, S. epidermis, S.
intermedias, Streptococcus hemolyticus, S. pneumoniae;
Gram-negative bacteria such as Flavobacterium meningosepticum,
Helicobacter pylori, Hemophilus pneumoniae, H. influenzae,
Klebsiella pneumonia, Neisseria gonorrhoeae, Pseudomonas
aeruginosa, Shigella dysenteria, Salmonella typhi, S. paratyphi,
Escherichia coli serotype 0157:H7, Chlamydia species; viruses such
as HIV-1, -2, and -3, HSV-I and -II, non-A non-B non-C hepatitis
virus, pox viruses, rabies viruses, and Newcastle disease virus;
fungi such as Candida albicans, C. tropicalis, C. krusei, C.
pseudotropicalis, C. parapsilosis, C. quillernondii, C.
stellatoidea, Aspergillus fumigatus, A. niger, A. nidulans, A.
flavus, A. terreus, Absidia corymbifera, A. ramosa, Cryptococcus
neoforms, Histoplasma capsulatum, Coccidioides immitis,
Pneumocystis carinii, Rhizopus arrhizus, R. oryzae, Mucor pusillus
and other fungi; and protozoa such as Entamoeba histolytica,
Entamoeba coli, Giardia lamblia, G. intestinalis, Eimeria sp.,
Toxoplasma sp., Cryptosporidium parvum, C. muris, C. baileyi, C.
meleagridis, C. wrairi, and C. nosarum. Obtaining unique epitopes
from these organisms by screening proteins and by assaying peptides
in vitro are commonly known to those skilled in the art.
[0070] The phrase "genetic defect" means any inheritable
pathological condition which is caused by the presence of a mutant
allele or disease gene. Examples include but are not limited to:
cystic fibrosis, Fabry disease, Gaucher disease, Tay-Sachs disease,
sickle cell anemia, Lesch-Nyhan disease, Parkinsons disease,
amyotrophic lateral sclerosis, Crohns disease, diabetes mellitus,
mannosidosis disease, celiac disease, X-linked glomerular disease,
and mucopolysaccharidosis.
[0071] Cross-Linking Agents
[0072] The term "attachment" with respect to an enzymatic substrate
and a first surface of a microcantilever, means a covalently bonded
or other physically connected molecule of substrate that is
connected to the coating material on the first surface of the
microcantilever. In a preferred embodiment, an attachment is a
covalent bond from the substrate to an atom of a chemical linker,
e.g., a bifunctional cross-linking reagent, which is also
covalently bonded through a different atom to the first surface.
Attachment can also be by direct non-covalent connection of the
biomaterial to the coating material on the first surface without
modification of either the first surface or to the biomolecule.
Such connection can be due to complementarity of shape, charge,
and/or to exclusion of waters of hydration, hydrophobicity, or
other characteristics of the of the particular combination of the
first surface and the particular substrate (U.S. Pat. No.
6,123,819, issued Sep. 26, 2000).
[0073] The phrase "bifunctional cross-linker" means a substance
which can connect a first component to a second component, wherein
the cross-linker consists of a carbon chain and has a first
chemically reactive group at a first end of the substance and a
second bioreactive group at a second end of the substance. A
chemical reaction between the first end of the substance with a
first component, and a chemical reaction between the second end of
the substance with a second component, results in the linkage of
the first and second embodiments of the invention herein, a
bifunctional cross-linker is used to bind a substrate molecule to a
first surface of a microcantilever, for example, to bind a protein
substrate such as a collagen to a first surface having a gold
coating.
[0074] For example, bifunctional cross-linkers can include the
following compounds: dithiobis(succinimidyl-undecanoate) (DSU), and
can be purchased from Pierce Endogen, Inc. (Rockford, Ill.); long
chain succinimido-6-[3-(2-pyridyldithio)-propionamido] hexanoate
(LCSPDP), contains pyridyldithio and NHS ester reactive groups
which react with sulfhydryl and amino groups, can be purchased from
Pierce; succinimidyl-6-[3-(2-pyridyldithio)propionamido] hexanoate
(SPDP) contains pyridyldithio and NHS ester reactive groups which
react with sulfhydryl and amino groups, can be purchased from
Pierce (Rockford, Ill.); and
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) contains NHS
ester and maleimide reactive groups which react with amino and
sulfhydryl groups, and can be purchased from Pierce (Rockford,
Ill.).
[0075] The terms "protein", "polypeptide", and "peptide", as used
herein, shall have the same meaning.
[0076] The above embodiments of the invention, having been fully
described, are illustrated by the following Examples, which are not
intended to be further limiting.
EXAMPLES
Example 1
Papain Digestion of an Immunoglobin IgG Antibody Substrate
[0077] A surface having a gold-coated microcantilever was cleaned
by exposure to an ozone-enriched atmosphere for 10 min. The
cross-linking agent was attached by immersing the microcantilever
in a solution of 0.1% (w/v) DSU in dioxane for 60 min. The
microcantilever was washed, for example, three times with dioxane,
followed by a wash with phosphate buffered saline (PBS), pH
7.6.
[0078] The microcantilever was further incubated with a solution of
Immunoglobulin G (1 mg/mL; CalBiochem, San Diego, Calif.) in PBS
solution for 60 min, to covalently attach a protein substrate for
the enzyme papain to the coated first of the microcantilever
surface. The microcantilever was removed from the antibody solution
and immersed in a carbonate buffer solution, pH 8.5, for 30 min to
hydrolyze any unreacted DSU.
[0079] The microcantilever was mounted in a cell of an AFM, and
measurement of microcantilever deflection was initiated. After
attainment of a stable baseline, a 100 microliter sample of a PBS
solution containing a 0.1% (w/v) solution of the detergent Tween
was injected into the cell. Microcantilever deflection was
monitored as a function of time, as is depicted in FIG. 4 as
"control." Next, a 100 microliter sample of papain (100 micrograms
per mL; CalBiochem, San Diego, Calif.) was injected into the cell.
Microcantilever deflection was monitored as a function of time, and
the results are depicted in FIG. 4, labeled as "papain."
[0080] The steady upward bending of the microcantilever shown in
FIG. 4 denotes loss of mass of the protein substrate from the
gold-coated first surface of the microcantilever. The data shown
are one example of several observations, having the same result.
The data show monitoring of enzymatic activity of papain as a
function of time. Further, these data show the capability of the
microcantilever to measure enzymatic activity.
Example 2
Neisseria Secreted Protease Digestion of IgG Substrate
[0081] A surface having a gold-coated microcantilever is cleaned by
exposure to an ozone-enriched atmosphere for 10 min. The
cross-linking agent is attached by immersing the microcantilever in
a solution of 0.1% (w/v) DSU in dioxane for 60 min. The
microcantilever is washed with dioxane, followed by a wash with
phosphate buffered saline (PBS), pH 7.6.
[0082] The microcantilever is further incubated with a solution of
Immunoglobulin G (1 mg/mL; CalBiochem, San Diego, Calif.) in PBS
solution for 60 min to covalently attach the IgG protein substrate
to the surface. The microcantilever is removed from the antibody
solution and immersed in a carbonate buffer solution, pH 8.5, for
30 min to hydrolyze any unreacted DSU.
[0083] The microcantilever is mounted in a cell of an AFM and
measurement of deflection is initiated. After attainment of a
stable baseline, a 100 microliter aliquot of a PBS solution
containing 0.1% (w/v) solution of the detergent Tween is injected
into the cell. Microcantilever deflection is monitored as a
function of time. Next, a 100 microliter aliquot of a sample
containing a Neisseria secreted protease is injected into the cell.
Microcantilever deflection is further monitored as a function of
time.
[0084] The steady upward bending of the microcantilever indicates
loss of mass of the protein substrate from the gold-coated side of
the microcantilever. Many other bacterial pathogens secrete a
similar antibody-specific proteolytic enzyme during a course of
pathogenesis, which enzyme can be detected by use of a
microcantilever.
* * * * *