U.S. patent application number 13/344315 was filed with the patent office on 2012-05-03 for immobilization particles for removal of microorganisms and/or chemicals.
This patent application is currently assigned to NUBIOME, INC.. Invention is credited to Brian C. Lue.
Application Number | 20120108787 13/344315 |
Document ID | / |
Family ID | 45997395 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120108787 |
Kind Code |
A1 |
Lue; Brian C. |
May 3, 2012 |
Immobilization Particles for Removal of Microorganisms and/or
Chemicals
Abstract
An immobilization particle for immobilizing a target
microorganism or target chemical found in or on a mammal that
includes: immobilization molecules capable of attaching to a target
microorganism or a target chemical, which immobilization molecules
are attached to one or more portions of a substrate structure;
wherein the substrate structure is capable of inhibiting contact
between tissues of the mammal and target microorganisms or target
chemicals attached to immobilization molecules attached to the one
or more portions.
Inventors: |
Lue; Brian C.; (Mountain
View, CA) |
Assignee: |
NUBIOME, INC.
Mountain View
CA
|
Family ID: |
45997395 |
Appl. No.: |
13/344315 |
Filed: |
January 5, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12660459 |
Feb 26, 2010 |
|
|
|
13344315 |
|
|
|
|
61212375 |
Apr 11, 2009 |
|
|
|
61208629 |
Feb 26, 2009 |
|
|
|
Current U.S.
Class: |
530/300 ;
530/391.1; 536/23.1 |
Current CPC
Class: |
C12Q 1/24 20130101; A61K
9/0031 20130101; C07K 16/08 20130101; A61K 9/7007 20130101; A61K
9/02 20130101; A61K 9/4841 20130101; C07K 16/14 20130101; G01N
33/569 20130101; A61K 31/713 20130101; C07K 16/12 20130101; A61K
9/0053 20130101; A61K 9/06 20130101 |
Class at
Publication: |
530/300 ;
530/391.1; 536/23.1 |
International
Class: |
C07K 17/00 20060101
C07K017/00; C07H 21/04 20060101 C07H021/04; C07H 21/02 20060101
C07H021/02 |
Claims
1. An immobilization particle for immobilizing a target
microorganism or target chemical found in or on a mammal that
comprises: immobilization molecules capable of attaching to the
target microorganism or the target chemical, which immobilization
molecules are attached to one or more portions of a substrate
structure; wherein the substrate structure is capable of inhibiting
contact between tissues of the mammal and target microorganisms or
target chemicals attached to immobilization molecules attached to
the one or more portions.
2. The immobilization particle of claim 1 wherein the
immobilization molecules are one or more of antibodies or
aptamers.
3. The immobilization particle of claim 2 wherein the substrate
structure comprises a substrate; and the one or more portions
comprise cavities in one or more sides of the substrate.
4. The immobilization particle of claim 2 wherein the substrate
structure comprises a substrate formed on a substrate support; and
the one or more portions are cavities in the substrate.
5. The immobilization particle of claim 2 wherein the substrate
structure comprises a substrate having spacers attached to one or
more sides of the substrate.
6. The immobilization particle of claim 5 wherein the spacers are
columns.
7. The immobilization particle of claim 2 wherein the aptamer is
one or more of a single-stranded RNA, a DNA molecule or a
peptide.
8. The immobilization particle of claim 2 wherein the substrate
structure comprises a gold substrate.
9. The immobilization particle of claim 8 wherein the substrate
structure comprises a gold substrate disposed on a polymer
substrate support.
10. The immobilization particle of claim 2 wherein the substrate
structure comprises a wall having an inner surface, which wall is
disposed on a substrate; and the one or more portions are disposed
on a surface of the substrate.
11. The immobilization particle of claim 2 wherein the substrate
structure comprises a wall having an inner surface disposed about
an open space; and the one or more portions are disposed on an
inner surface of the wall.
12. The immobilization particle of claim 11 wherein the wall
includes an opening.
13. The immobilization particle of claim 2 wherein the substrate
structure comprises a polymer substrate.
14. The immobilization particle of claim 2 wherein the substrate
structure comprises a substrate; and the substrate includes one or
more pores in one or more sides of the substrate.
15. The immobilization particle of claim 14 wherein the one or more
portions comprise one or more of the one or more pores.
16. The immobilization particle of claim 15 which further comprises
a substrate support abutted to the substrate.
17. The immobilization particle of claim 16 which further comprises
spacers attached to one or more sides of the substrate.
Description
[0001] This patent application is a continuation-in-part of a U.S.
patent application having application Ser. No. 12/660,459 filed on
Feb. 26, 2010 and that claimed priority under 35 U.S.C. 119(e) from
a U.S. provisional application having Appl. No. 61/208,629 filed
Feb. 26, 2009 and a U.S. provisional application having Appl. No.
61/212,375 filed Apr. 11, 2009, all of which patent applications
are incorporated herein in their entireties.
TECHNICAL FIELD
[0002] One or more embodiments relate to apparatus for use in
removing microorganisms of chemicals.
BACKGROUND
[0003] The mixture of microorganisms in a person's gastrointestinal
tract greatly affects the person's health. Some beneficial effects
provided by the mixture of microorganisms are: aiding in food
digestion, creating vitamins, sequestering and neutralizing toxic
metals, creating anti-cancer compounds, secreting beneficial
enzymes, and preventing pathogenic microorganisms from colonizing
the gastrointestinal tract.
[0004] From the time a person is approximately one year old until
s/he is in her/his 50's to 60's, the composition of the mixture of
microorganisms, for example, bacteria, and the population thereof
is mostly stable. A combination of genetics, bacterial exposure
from the environment and a person's diet help determine the strains
and quantities of bacteria that colonize the person's
gastrointestinal tract. For most normal, healthy individuals, their
microbial population, or microbiome, does not cause any problems.
Unfortunately, for others, their microbiome becomes dysfunctional
and creates various chronic health problems.
[0005] There are many triggers that cause a microbiome to become
dysfunctional. One common trigger is the use of antibiotics and
antifungals. Antibiotics and antifungals kill many kinds of
bacteria and fungi, both helpful and harmful. When antibiotics
and/or antifungals are taken into the body, beneficial bystander
bacteria and/or fungi (i.e. bacteria or fungi that are not the
intended target of the antibiotics or antifungals) get killed. As a
result, the natural balance of microorganisms in the microbiome may
be perturbed, and remaining beneficial bacteria and/or fungi can
lose their ability to inhibit harmful ones. In addition, certain
antibiotics can change the behavior of normally present bacteria
and make them harmful or more difficult for the immune system or
antibiotics to target. For example, Penicillin G makes Proteus
bacteria become cell wall deficient, and as a result, many
antibiotics cannot kill them.
[0006] Once a bacterial and/or fungal population is perturbed by
antibiotics and/or antifungals, enzymes present in the
gastrointestinal tract can change and the normal distribution of
peptides seen by the immune system can change. If peptide sequences
that sufficiently resemble various molecules of a host's organ or
other tissue survive in sufficient concentrations, autoimmune
disease may result.
SUMMARY
[0007] One or more embodiments solve one or more of the
above-identified problems. In particular, one embodiment is an
immobilization particle for immobilizing a target microorganism or
target chemical found in or on a mammal that comprises:
immobilization molecules capable of attaching to a target
microorganism or a target chemical, which immobilization molecules
are attached to one or more portions of a substrate structure;
wherein the substrate structure is capable of inhibiting contact
between tissues of the mammal and target microorganisms or target
chemicals attached to immobilization molecules attached to the one
or more portions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows a perspective view of an immobilization
particle that is fabricated in accordance with one or more
embodiments of the present invention, and
[0009] FIG. 2 shows a side view of the immobilization particle
shown in FIG. 1.
[0010] FIG. 3 shows a perspective view of an immobilization
particle that is fabricated in accordance with one or more further
embodiments, and
[0011] FIG. 4 shows a side view of the immobilization particle
shown in FIG. 3.
[0012] FIG. 5 shows a perspective view of an immobilization
particle that is fabricated in accordance with one or more still
further embodiments, and
[0013] FIG. 6 is a side view of the immobilization particle shown
in FIG. 5.
[0014] FIG. 7 shows a perspective view of an immobilization
particle that is fabricated in accordance with one or more still
further embodiments.
[0015] FIG. 8 shows a perspective view of an immobilization
particle fabricated as an array of the immobilization particle
shown in FIG. 7.
[0016] FIG. 9 shows a perspective view of an immobilization
particle that is fabricated in accordance with one or more still
further embodiments.
[0017] FIG. 10 shows a perspective view of an immobilization
particle that is fabricated in accordance with one or more still
further embodiments.
[0018] FIG. 11 shows a perspective view of an immobilization
particle that is fabricated in accordance with one or more still
further embodiments.
[0019] FIG. 12 shows a perspective view of an immobilization
particle that is fabricated in accordance with one or more still
further embodiments.
[0020] FIG. 13 shows a cross-section of an immobilization particle
that is fabricated in accordance with one or more still further
embodiments.
DETAILED DESCRIPTION
[0021] One or more embodiments of this invention are immobilization
particles that immobilize microbes and/or chemicals found, for
example and without limitation, in a human's or in an animal's
gastrointestinal tract. In accordance with one or more further
embodiments of the present invention, such immobilization particles
that are introduced into the gastrointestinal tract may be removed
by natural processes and/or as aided by mechanical processes such
as, but not limited to, enema, suction, or magnetic attraction.
[0022] In accordance with one or more embodiments of the present
invention, a target microorganism and/or chemical becomes attached
to an immobilization particle, and as a result, the target
microorganism or chemical can be removed from the body. In
accordance with one or more further embodiments, immobilization
particle is constructed so the attached target microorganism and/or
chemical does not physically interact with gastrointestinal tract
tissue. In accordance with one or more such embodiments, for use of
an immobilization particle to attach to a microorganism (as used
herein, the terms microorganism and microbe are the same), the
immobilization particle can remove the microbe without killing it
and without the microbe breaking up into pieces. Advantageously,
keeping a microbe whole and out of physical contact with a host's
gastrointestinal tract tissue may help prevent an undesirable
interaction with the host's immune system, which interaction may
lead to harmful inflammation. In addition and in accordance with
one or more further such embodiments of the present invention, for
the case of a piece of a microorganism that could be
immunologically active, the immobilization particle can remove the
piece of microorganism before it causes unwanted reactions in the
body. In addition and in accordance with one or more further such
embodiments of the present invention, for the case of the use of an
immobilization particle to attach to a chemical, the immobilization
particle can remove the chemical before it causes unwanted
reactions. Such unwanted reactions may, for example and without
limitation, produce concentrations enzymes that, in turn, can cause
abnormal concentrations of peptide sequences which, in turn, may
cause an autoimmune reaction. As such, an immobilization particle
that is fabricated in accordance with one or more embodiments of
the present invention may be used to remove triggers autoimmune
disease and its symptoms, which triggers, for example and without
limitation, may be peptide sequences that resemble portions of
human tissue.
[0023] In accordance with one or more embodiments of the present
invention, an immobilization particle comprises a substrate
structure comprised of a substrate that includes one or more
concave surfaces, one or more recesses, and/or one or more pores.
In accordance with one or more such embodiments, an immobilization
particle further comprises immobilizing molecules attached to at
least a portion of the surface of the substrate. In accordance with
one or more further embodiments of the present invention, an
immobilization particle comprises a substrate structure wherein
spacer structures are affixed to the substrate and wherein the
spacer structures are adapted to inhibit contact between the
immobilization molecules and tissue such as, for example and
without limitation, tissue or mucosa of the gastrointestinal wall.
As such, in providing spacer structures, one would take into
account the size of immobilization molecules and a size of a target
microorganism or a chemical. In accordance with one or more such
embodiments, the substrate structure further comprises a substrate
support with is attached to the substrate, wherein the spacer
structures are affixed to the substrate or to the substrate
support.
[0024] FIG. 1 shows a perspective view of immobilization particle
100 that is fabricated in accordance with one or more embodiments,
and FIG. 2 shows a side view of immobilization particle 100. As
shown in FIG. 1, immobilization particle 100 includes (a) a
substrate structure comprised of substrate 101 and spacers 102; and
(b) immobilization molecules 103. As shown in FIG. 1, substrate 101
is planar and spacers 102 and immobilization molecules 103 are
attached to substrate 101. Spacers 102 are sufficiently tall, and
spaced closely enough together so that, in a particular
application, they are capable of inhibiting physical contact
between tissue, for example and without limitation, tissue from a
flexible gastrointestinal wall and/or dendritic cells attached to
the gastrointestinal wall and target microorganisms and/or
chemicals attached to immobilization molecules 103. In operation,
the top surfaces of spacers 102 push the host's tissues away from
surfaces of substrate 101 where the immobilization molecules are
attached. As shown in FIG. 2, spacers 102 have a height H and a
width D, the distance between the centers of spacers 102 is P, and
the distance between immobilization particles 103 and the top of
spacers 102 is G. For example, H is large enough so that a target
microorganism or chemical can attach to immobilization molecules
103 without the target microorganism or target chemical coming into
physical contact with the host's tissues. Appropriate height and/or
spacing for spacers 102 for a particular application can be
determined routinely by one of ordinary skill in the art without
using undue experimentation. For example, appropriate height and
spacing depends, among other things on the particular
immobilization molecule, and the targeted microorganism and/or
chemical, and the particular tissue whose touch is to be inhibited
or avoided. In some applications, the taller spacers 102 are, the
farther apart they can be. It should be understood by those of
ordinary skill in the art that further embodiments exist where the
heights of spacers 102 may be different, and/or the distances
between spacers 102 may be different, and/or immobilization
molecules 103 may be different. In accordance with one or more
embodiments, the height of spacers is in a range from about 0.01
microns to about 500 microns and the distance between spacers is in
a range from about 0.1 microns to about 500 microns.
[0025] In accordance with one or more embodiments, substrate 101
and spacers 102 can be made of, for example and without limitation,
one or more of the following materials: polymer, metal, ceramic,
semiconductor, carbohydrate, polysaccharide, polypeptide, protein,
glycolipid, gel, highly viscous glass or a combination or composite
of one or more of the foregoing. Examples of suitable polymers
include, for example and without limitation, one or more of
silicone, polyethylene, polystyrene, polyurethane,
polymethacrylate, polyester, and polycarbonate. In accordance with
one or more further embodiments of the present invention, the
substrate and spacers are comprised of a polymer film such as, for
example and without limitation, Mylar or a woven or compressed
polyester fabric. The materials can be molded, chemical vapor
deposited, physical vapor deposited, ground, etched, extruded,
solution precipitated, blown, vapor phase reacted, crushed,
tumbled, polished, chemical mechanical planarized, electro
discharge machined, pressed, stamped, lased, machined, poured,
spun, pulled, pressed, welded, bonded, diffusion bonded, friction
bonded, ultrasonically welded, ion beam welded, ion beam deposited,
punched, pressure formed, gouged, cut, laser cut, abrasive blasted,
freeze fractured, chemically foamed and cooled, cured, UV cured,
photo-lithographed, 3D printed, stereo-lithographed, silk-screened,
ink jet printed, fused, or made using any combination of the
previously mentioned processes, which processes are well known to
those of ordinary skill in the art.
[0026] FIG. 3 shows a perspective view of immobilization particle
200 that is fabricated in accordance with one or more further
embodiments, and FIG. 4 shows a side view of immobilization
particle 200. As shown in FIG. 3, immobilization particle 200
includes (a) a substrate structure comprised of substrate 201,
planar substrate support 202 and spacers 203; and (b)
immobilization molecules 203. As shown in FIG. 3, substrate 201 is
planar. As indicated in FIG. 3, spacers 203 are attached to
substrate support 202, and immobilization molecules 204 are
attached to substrate 201. It should be understood by those of
ordinary skill in the art that further embodiments exist where
spacers 203 are attached to substrate 201. Spacers 203 are
sufficiently tall, and spaced closely enough together, so that, in
a particular application, they are capable of inhibiting physical
contact between tissue, for example and without limitation, tissue
from a flexible gastrointestinal wall and/or dendritic cells
attached to the gastrointestinal wall and microorganisms and/or
chemicals attached to immobilization molecules 204. In operation,
the top surfaces of spacers 203 push the host's tissues away from
surfaces of substrate 201 where immobilization molecules 204 are
attached. As shown in FIG. 4, spacers 203 have a height H and a
width D, the distance between the centers of spacers 203 is P, and
the distance between immobilization particles 204 and the top of
spacers 203 is G. For example, H is large enough so that a target
microorganism or chemical can attach to immobilization molecules
204 without the target microorganism or target chemical coming into
contact with the host's tissues. Appropriate height and/or spacing
for spacers 203 for a particular application can be determined
routinely by one of ordinary skill in the art without using undue
experimentation. For example, appropriate height and spacing
depends, among other things on the particular immobilization
molecule, and the targeted microorganism and/or chemical, and the
particular tissue whose touch is to be inhibited or avoided. It
should be understood by those of ordinary skill in the art that
further embodiments exist where the heights of spacers 203 are
different, and/or the distances between spacers 203 are different,
and/or immobilization molecules 204 are different. In accordance
with one or more embodiments, the height of spacers is in a range
from about 0.01 microns to about 500 microns and the distance
between spacers is in a range from about 0.1 microns to about 500
microns.
[0027] In accordance with one or more such embodiments, substrate
201 is attached to substrate support 202, and substrate support 202
is thick enough to enable substrate 201 to maintain a predetermined
shape. In accordance with one or more such embodiments, substrate
201 and substrate support 202 may be fabricated from the same
materials set forth above for substrate 101, spacers 203 may be
fabricated from the same materials set forth above for spacers 102,
and substrate 201, substrate support 202 and spacers 203 may be
fabricated using the same methods set forth above for substrate 101
and spacers 102. In accordance with one or more such embodiments,
substrate 201 and substrate support 202 can be the same material or
a different material, also, substrate 201 may be a coating which is
deposited on substrate support 202.
[0028] FIG. 5 shows a perspective view of immobilization particle
300 that is fabricated in accordance with one or more still further
embodiments, and FIG. 6 is a side view of the immobilization
particle shown in FIG. 5. As shown in FIG. 5, immobilization
particle 300 includes (a) a substrate structure comprised of
substrate 302 and spacer 303; and (b) immobilization molecules 301.
As shown in FIG. 5, substrate 302 is planar and spacer 303 and
immobilization molecules 301 are attached to substrate 302. As
shown in FIG. 5, spacer 303 is formed as a wall that surrounds a
predetermined area of substrate 302. The walls of spacer 303 are
sufficiently tall, and are spaced closely enough together so that,
in a particular application, they are capable of inhibiting
physical contact between tissue, for example and without
limitation, tissue from a flexible gastrointestinal wall and/or
dendritic cells attached to the gastrointestinal wall and target
microorganisms and/or chemicals attached to immobilization
molecules 301. In operation, top surfaces 304 of spacer 303 push
the host's tissues away from surface 305 of substrate 302 where
immobilization molecules 301 are attached. Although the walls of
spacer 303 form a square, it should be understood by those of
ordinary skill in the art that further embodiments exist where the
walls of spacer 303 form a rectangle and other embodiments exist
where the walls of spacer 303 form a polygon such as a triangle, or
even form a curvilinear shape such as, for example and without
limitation, a cylinder. As shown in FIG. 6, the walls of spacer 303
have a height H, a width K, the distance between the outer edges of
the walls is W, and the distance between the inner edges of the
walls is J, and the distance between immobilization particles 301
and the top surface of the walls is G. For example, H is large
enough and J is small enough so that a target microorganism or
target chemical can attach to immobilization molecules 301 without
the target microorganism or target chemical coming into contact
with the host's tissues. Appropriate height and/or spacing for the
walls of spacer 303 for a particular application can be determined
routinely by one of ordinary skill in the art without using undue
experimentation. For example, appropriate height and spacing
depends, among other things on the particular immobilization
molecule, and the targeted microorganism and/or chemical, and the
particular tissue whose touch is to be inhibited or avoided. In
accordance with one or more embodiments, the height of walls is in
a range from about 0.01 microns to about 500 microns and the
distance between walls is in a range from about 0.1 microns to
about 500 microns. It should be understood by those of ordinary
skill in the art that further embodiments exist where an
immobilization particle comprises several units like that shown in
FIGS. 5 and 6, and that in such further embodiments the heights of
the walls of the various spacers may be different, and/or the
shapes of the walls of the spacers may be different, and/or
immobilization molecules 301 in the various units may be different.
In addition, further embodiments exist where the walls of spacer
303 are not continuous, but are comprised of discontinuous
segments. Still further embodiments exist where the walls of spacer
303 and the perimeter of substrate 302 are not aligned and where
the perimeter of substrate 302 is not rectangular. Yet still
further embodiments exist where the height H of top surface 304 of
the walls of spacer 303 vary--for example and without limitation,
top surface 304 may a wavy surface.
[0029] As one of ordinary skill in the art can readily appreciate,
by attaching spacer 303 to substrate 302, a concave-shaped
immobilization particle is formed such that surface 305 of
substrate 302 may be viewed as an inner concave surface of
immobilization particle 300. The interior surfaces of concave
cavities are preferred surfaces to which immobilization molecules
are attached. This is because targets, such as, for example and
without limitation, single celled organisms or parts of organisms,
captured by immobilization molecules attached to interior surfaces
of a concave cavity are shielded from physical contact with
surfaces of a host's gastro-intestinal wall by the concave cavity.
In addition, the concave cavity makes it more difficult for
tentacles of dendritic cells in the host's gastro-intestinal wall
to reach targets captured within the concave cavity. It is believed
that preventing physical contact with the target is beneficial in
that it prevents a host's immune system from becoming aware of the
target's presence. This, in turn, prevents the host's immune system
from generating an immune response by generating cross-reacting
antibodies to the immobilized targets, which antibodies could, in
the case of autoimmunity, attack other body tissues.
[0030] In accordance with one or more such embodiments, substrate
302 and spacer 303 may be fabricated from the same materials set
forth above for substrate 101, and substrate 302 and spacers 303
may be fabricated using the same methods set forth above for
substrate 101 and spacers 102. It should also be understood that
further embodiments exist where the substrate structure includes a
substrate support that is fabricated in the same manner described
above with respect to immobilization particle 200.
[0031] FIG. 7 shows a perspective view of immobilization particle
400 that is fabricated in accordance with one or more further
embodiments. As shown in FIG. 7, immobilization particle 400
includes (a) a substrate structure comprised of substrate 402 which
includes a concavity, with concave surface 403, within substrate
402; and (b) immobilization molecules 401 attached to concave
surface 403. As shown in FIG. 7, a surface of substrate 402
surrounding the opening of the concavity is substantially planar,
and the shape of the opening of the concavity is substantially
circular. In accordance with one or more such embodiments, the
depth at which immobilization molecules 401 are attached to concave
surface 403 (as measured from the surface of substrate 402 that
surrounds the opening of the concavity) is sufficiently large so
that, in a particular application, tissue, for example and without
limitation, tissue from a flexible gastrointestinal wall and/or
dendritic cells attached to the gastrointestinal wall is capable of
being inhibited from making physical contact with target
microorganisms and/or chemicals attached to immobilization
molecules 401. In operation, immobilization top surfaces of
substrate 402 surrounding the cavity push the host's tissues away
from concave surface 403 where immobilization molecules 401 are
attached. In accordance with one or more embodiments, concave
surface 403 has a depth in a range from about 0.02 microns to about
3000 microns, and immobilization molecules are affixed at depths in
a range from about 0.01 microns to about 3000 microns.
[0032] In accordance with one or more such embodiments, substrate
402 may be fabricated from the same materials set forth above for
substrate 101. It should also be understood that further
embodiments exist where the substrate structure includes a
substrate support disposed on substrate 402 in the manner described
above with respect to immobilization particle 200. It should also
be understood that further embodiments exist where the opening of
the concavity in substrate 402 has a non-circular shape such as,
for example and without limitation, an elliptical shape, the shape
of a kidney bean, or other shapes. It should also be understood
that further embodiments exist wherein the surface of the opening
of the concavity in substrate 402 is non-planar.
[0033] FIG. 8 shows a perspective view of immobilization particle
500 that is fabricated in accordance with one or more further
embodiments. As shown in FIG. 8, immobilization particle 500
includes a substrate structure comprised of substrate 504 which
includes an array of concavities disposed so that openings of the
concavities are alternately disposed at a top surface and a bottom
surface of substrate 504, respectively. In addition, as shown in
FIG. 8, the concavities are formed so that they provide a
convexities in substrate 504 on a surface of 504 opposite from
their openings. In accordance with one or more such embodiments,
one or more such concavities is like the concavity described above
with respect to immobilization particle 500. As such, as shown in
FIG. 8, immobilization molecules 501 are attached to concave
surface 503. Although not shown in FIG. 8, in accordance with one
or more embodiments, immobilization molecules may be attached to
concave surfaces 503 at any depth with respect to the opening
thereof, as well as, at planar portions of substrate 504. In
accordance with one or more such embodiments, convex surfaces, like
convex surface 502 of an adjacent concavity in substrate 504
functions as a spacer. In accordance with one or more such
embodiments, the height of convex surfaces 502 above planar
portions of substrate 504 are sufficiently tall, and are spaced
closely enough together, so that, in a particular application,
convex surfaces 502 are capable of inhibiting physical contact
between tissue, for example and without limitation, tissue from
flexible gastrointestinal wall and/or dendritic cells attached to
the gastrointestinal wall and microorganisms and/or chemicals
attached to immobilization molecules 501. In operation, convex
surface 502 pushes the host's tissues away from surfaces 503 and
504 where immobilization molecules 501 are attached. Although
convex surfaces 502 and concave surfaces 503 are shown in FIG. 8 as
tapered conical surfaces, it should be understood by those of
ordinary skill in the art that further embodiments exist where
surfaces 503 and 502 are polygonal or even have a rectilinear shape
such as, for example and without limitation, a box or pyramidal
shape. A depth of concave surface 503 should be sufficiently deep
so that target microorganisms and/or chemicals can attach to
immobilization molecules 501 without coming into contact with the
host's tissues. Appropriate height and/or spacing for concave
surfaces 503 and convex surfaces 502 for a particular application
can be determined routinely by one of ordinary skill in the art
without using undue experimentation. For example, appropriate
height and spacing depends, among other things on the particular
immobilization molecule, the targeted microorganism and/or
chemical, and the particular tissue whose touch is to be inhibited
or avoided. In accordance with one or more embodiments, the depths
of the concavities and convexities are in a range from about 0.02
microns to about 3000 microns, and a distance between adjacent
concavities and convexities is in a range from about 0.1 microns to
about 5000 microns. It should be understood by those of ordinary
skill in the art that further embodiments exist where an
immobilization particle is attached to concave surfaces and the
depths of the concave surfaces and heights of the convex surfaces
are different, and/or the shapes of concave surfaces 503 and convex
surfaces 502 are different. In addition, further embodiments exist
where the spacing between repeated concave and convex surfaces is
irregular and/or do not line up in a rectilinear fashion.
[0034] FIG. 9 shows a perspective view of immobilization particle
600 that is fabricated in accordance with one or more further
embodiments. As shown in FIG. 9, immobilization particle 600 is the
same as immobilization particle 400 described above in conjunction
with FIG. 7 except that the concavity of immobilization particle
600 has a shape like that of immobilization particle 300 described
above in conjunction with FIG. 5. As shown in FIG. 9,
immobilization molecules are not attached to flange 601 which
surrounds wall 603. It should be understood that further
embodiments exist where the substrate structure of immobilization
particle 600 includes a substrate support in the same manner
described above with respect to the substrate support of
immobilization particle 200.
[0035] FIG. 10 shows a perspective view of immobilization particle
700 that is fabricated in accordance with one or more further
embodiments. As shown in FIG. 10, immobilization particle 700
includes (a) a substrate structure comprised of wall 703 that
surrounds hollow space 704; and (b) immobilization molecules 701
attached to inner concave surfaces 702 of wall 703. As further
shown in FIG. 10, the substrate structure of immobilization
particle 700 has open ends that enable target microorganisms and/or
chemicals to enter hollow space 704 where they become attached to
immobilization molecules 701. In accordance with one or more such
embodiments, immobilization molecules 701 are attached sufficiently
far from the open ends of the hollow space 704 so that attached
target microorganisms and/or chemicals do not interact with a
host's tissues as discussed above. Although FIG. 10 shows wall 703
having four interior sides which form a closed structure
surrounding hollow space 704, it should be understood that further
embodiments exist where wall 703 has three or more sides that form
a polygonal wall structure about hollow space 704. In addition, in
still further embodiments, the sides of the wall may be curvilinear
instead of being planar. In addition, yet still further embodiments
exist where a substrate support may be attached to outer surfaces
of the sides of wall 703. In accordance with one or more such
embodiments, immobilization particle 700 may be fabricated from the
same materials set forth above for immobilization particle 300.
[0036] FIG. 11 shows a perspective view of immobilization particle
800 that is fabricated in accordance with one or more further
embodiments. As shown in FIG. 11, immobilization particle 800 is
the same as immobilization particle described above in conjunction
with FIG. 10 except that wall 804 includes slot 801. Slot 801
enables target microorganisms and/or chemicals to enter the hollow
space and attach to immobilization molecules 802. The width of slot
801 is sufficient to permit the entrance of target microorganisms
and/or chemicals. It should be understood that embodiments exist
where slot 801 does not extend along the entire length of wall 804.
Appropriate width and length for slot 801 for a particular
application can be determined routinely by one of ordinary skill in
the art without using undue experimentation. In accordance with one
or more embodiments, slot 801 has a width in a range from about 0.5
microns to about 100 microns, and a length in a range from about 1
micron to about 5000 microns.
[0037] FIG. 12 shows a perspective view of immobilization particle
900 that is fabricated in accordance with one or more further
embodiments. As shown in FIG. 12, immobilization particle 900
includes (a) a substrate structure comprised of multiple, spaced
units, which units include substrate 903, substrate support 904 and
spacers 901; and (b) immobilization molecules 902. Spacers 901 are
sufficiently tall to permit the entrance of target microorganisms
and/or chemicals into the units where they become attached to
immobilization molecules 902. Substrate supports 904 are
sufficiently thick to enable substrates 903 to maintain a
predetermined shape. Appropriate height for spacers 901 and
thickness for substrate supports 904 for a particular application
can be determined routinely by one of ordinary skill in the art
without using undue experimentation. In accordance with one or more
such embodiments, the height of spacers 901 is in a range from
about 0.01 micron to about 500 microns and the thickness of
substrate supports 904 is in a range from about 1 micron to about
500 microns. In accordance with one or more such embodiments,
spacers 901, substrates 903, and substrate supports 904 may be
fabricated from the same materials set forth above for spacers 203,
substrate 201, and substrate support 202. It should also be
understood by those of ordinary skill in the art that further
embodiments exist where substrates 903 are attached to both sides
of substrate supports 904, and that this enables immobilization
molecules 902 to be attached to substrates on either side of a gap
created by spacers 901.
[0038] FIG. 13 shows a cross-section view of immobilization
particle 1000 that is fabricated in accordance with one or more
further embodiments of the present invention. As shown in FIG. 13,
immobilization particle 1000 includes (a) a substrate structure
comprised of substrate 1001 and spacers 1003; and (b)
immobilization molecules 1002. Substrate 1001 is substantially
planar; immobilization molecules 1002 are attached to both sides of
substrate 1001; and spacers 1003 are attached to both sides of
substrate 1001. Having immobilization molecules 1002 and spacers
1003 attached to both sides of substrate 1001 simplifies
manufacturing because substrate 1001 can be completely immersed
while attaching immobilization molecules 1002. In accordance with
one or more such embodiments, substrate 1001 and spacer 1003 may be
fabricated from the same materials set forth above for spacers 102,
and substrate 1001 and spacers 1003 may be fabricated using the
same methods set forth above for substrate 102 and spacers 103. It
should also be understood that further embodiments exist where the
substrate structure of immobilization particle 1000 includes a
substrate support in the same manner described above with respect
to immobilization particle 200. Appropriate height and spacing of
spacers 1003 depends, among other things, on the particular
immobilization molecule, the targeted microorganism and/or
chemical, and the particular tissue whose touch is to be inhibited
or avoided. It should be understood by those of ordinary skill in
the art that further embodiments exist where an immobilization
particle comprises several units like that shown in FIGS. 5 and 6,
and that in such further embodiments the heights of the walls of
the various spacers may be different, and/or the shapes of the
walls of the spacers may be different, and/or immobilization
molecules 1002 in the various units may be different.
[0039] Immobilization Molecules
[0040] In accordance with one or more embodiments of the present
invention, an immobilization molecule is, for example and without
limitation, an antibody or an aptamer. Antibodies are molecules
that are produced by an immune system that attach specifically to
microorganisms and chemicals (microorganisms can also produce
antibodies). Antibodies are fairly large proteins (for example, a
typical protein weighs approximately 150 kDa), and suitable
antibodies can be created which are capable of binding to specific
proteins or specific chemicals. Antibodies used to fabricate one or
more embodiments of the present invention may be produced using
standard monoclonal or polyclonal antibody production techniques
that are well known to those of ordinary skill in the art. Aptamers
are synthetic molecules that can attach to microorganisms and
chemicals with high specificity. Suitable aptamers can be
chemically synthesized bits of single-stranded RNA or DNA molecules
or peptides whose selection is optimized by sorting processes that
are well known to those of ordinary skill in the art. An example of
such a sorting process is, but not limited to, a sorting process
referred to as "Systematic Evolution of Ligands by Exponential
Enrichment (SELEX)" in an article by Tuerk, et al. entitled
"Systematic evolution of ligands by exponential enrichment: RNA
ligands to bacteriophage T4 DNA polymerase." in Science, 3 Aug.
1990, 249, pp. 505-510. Aptamers are typically 100 nucleotides
long. In accordance with one or more embodiments, a particular
antibody and/or aptamer is selected for use with an intended target
microorganism or chemical.
[0041] In accordance with one or more such embodiments,
immobilization molecules can be attached to a substrate structure
by adsorption or by covalent linking. For adsorbing an
immobilization molecule to a substrate structure, one option is to
place the substrate structure into a liquid containing an aptamer
or antibody for a time long enough for the aptamer or antibody to
be adsorbed onto the substrate structure. For proper adsorption,
the substrate structure needs to be clean enough so that adsorption
occurs, which level of cleanliness may be determined readily by one
of ordinary skill in the art without undue experimentation.
[0042] Antibody Selection/Design--For Immobilizing Bacteria, Fungi,
and Viruses: In accordance with one or more embodiments of the
present invention, immobilization molecule(s) are antibodies and/or
their fragment antigen binding, which fragment antigen binding is a
region of the antibody that binds to an antigen. In accordance with
one or more such embodiments, the immobilization molecules are
antibodies and/or their fragment antigen binding that are useful to
immobilize, for example and without limitation, one or more of the
following genera of bacteria (use of such immobilization particles
provides a method of treating or mitigating symptoms of diseases
associated with such genera of bacteria): (a) for Multiple
Sclerosis, the following genera of bacteria, Enterococcus,
Streptococcus, Lactobacillus, Bacteroides, Escherichia,
Clostridium, Serratia, Bifidobacterium and Fusobacterium; (b) for
ulcerative colitis, the following genera of bacteria, Burkholderia,
Mycobacterium, Bacillus, Clostridium and Methylobacterium; (c) for
Lupus, the following genera of bacteria, Burkholderia,
Mycobacterium, Pseudomonas, Methylobacterium, Vibrio and
Clostridium; (d) for Uveoretinitis, the following genera of
bacteria, Bacteriodes, Bacillus, Clostridium, Lactobacillus,
Fusobacterium, Vibrio, Ruminococcus and Methylococcus; and (e) for
rheumatoid arthritis, the following genera of bacteria, gram
positive bacteria. It is believed that removing these genera of
bacteria from a host is useful as they can exacerbate symptoms
corresponding to the identified diseases. Suitable antibodies which
can serve as immobilization molecules to immobilize the
above-identified genera of bacteria are readily commercially
available, for example and without limitation, BacTrace Anti-Vibrio
Genus Antibody is a suitable antibody that is available from
Kirkegaard & Perry Laboratories, Inc. (accessible at
http://www.kpl.com).
[0043] In accordance with one or more further embodiments of the
present invention, antibodies and/or their fragment antigen binding
are selected to immobilize, for example and without limitation, one
or more of the following genera of fungi: Saccharomyces and
Candida. Suitable antibodies which can serve as immobilization
molecules to immobilize the above-identified genera of fungi are
readily commercially available, for example and without limitation,
Candida albicans (BGN/03/5424) Species Antibody is a suitable
antibody that is available from Santa Cruz Biotechnology, Inc.
(accessible at http://www.scbt.com).
[0044] In accordance with one or more further embodiments of the
present invention, antibodies and/or their fragment antigen binding
are selected/designed to immobilize, for example and without
limitation, one or more of the following viruses: Influenza, Herpes
and Cytomegalovirus. Suitable antibodies which can serve as
immobilization molecules to immobilize the above-identified genera
of viruses are readily commercially available, for example and
without limitation, Influenza A ml (156-02) Antibody is a suitable
antibody that is available from Santa Cruz Biotechnology, Inc.
(accessible at http://www.scbt.com).
[0045] Antibody Adsorption onto Polymer: In accordance with one or
more embodiments of the present invention, antibodies are attached
to a polymer substrate structure by adsorption using the following
steps. First, purify the antibody. Next, carry out an optional acid
pretreatment of the antibody. Next, clean the substrate structure
as described below. Next, adsorb the antibody onto the polymer
substrate structure as described below.
[0046] Purification of the Antibody: In accordance with one or more
embodiments of the present invention, antibodies and/or their
fragment antigen binding are purified using, for example and
without limitation, the following method: sodium sulphate
precipitation (20% w/v) followed by Sephacryl S-200 HR gel
filtration or protein A affinity chromatography, which method is
disclosed in an article by van Erp entitled "Monoclonal antibodies
in diagnostics. Monitoring of monoclonal antibody characteristics
during (large scale) production, purification and application in
diagnostic systems." Ph.D. Thesis, University of Nijmegen,
Nijmegen, Netherlands, 1991 available at the University of Nijmegen
Library, Nijmegen, Netherlands, and an article by van Erp et al.
entitled "Affinity of monoclonal antibodies: Interpretation of the
positive cooperative nature of anti-hCG/hCG interactions. J.
Immunol. Methods, 140, 1991, pp. 235-241.
[0047] Acid Pretreatment of Antibody: As disclosed in an article by
van Erp et al. entitled "Characterization of monoclonal antibodies
physically adsorbed onto polystyrene latex particles" in J. of
Immunol. Methods, 152 pp. 191-199, 1992, pre-treating antibodies
with hydrochloric acid, or an acid solution with a pH of
approximately 1.0-3.0, can improve the binding capacity of
antibodies to substrate structures. In accordance with one or more
embodiments of the present invention a pretreatment comprises, for
example and without limitation: (a) mixing antibodies with 0.05M
glycine/HCl buffer pH 2.0; (b) incubating the antibody solution at
0-4 degrees Celsius for 1 hour; and (c) adjusting the pH of the
mixture to 6.0-8.0 by the addition of 0.1 M NaOH.
[0048] Cleaning Polymer Substrate structures: In accordance with
one or more embodiments of the present invention, substrate 101,
substrate 201 and substrate 302 described above may be a polymer
such as, for example and without limitation, polystyrene, polyester
or nylon, and may require cleaning. Top surface 105 of substrate
101, top surface 206 of substrate 201 and top surface 305 of
substrate 302 can be cleaned using, for example and without
limitation, a cleaning solution made as follows. Prepare a
phosphate buffer by mixing phosphate buffer powder (for example,
phosphate buffer powder obtainable from Wako Pure Chemical of
Osaka, Japan) with ultra-pure water until a 1/15M solution having a
pH of 7.4 is achieved. Top surfaces 105, 206 and 305 of substrates
101, 201 and 302, respectively, may be cleaned by a method
disclosed in an article by Sato et al. entitled "Integration of an
Immunosorbant Assay System Analysis of Secretory Human
Immunoglobulin A on Polystyrene Beads in a Microchip" in Anal
Chem., 72, 2000, pp. 1144-1147, which method comprises: (a)
irrigating the top surfaces with the phosphate buffer solution; (b)
then rinsing with ultra-pure and/or deionized water; and (c) then
drying the surface.
[0049] It is useful to attach enough antibodies to the substrate
per unit surface area of substrate to immobilize a target
microorganism and/or chemical. There are several classes of
antibodies: IgA, IgD, IgE, IgG, and IgM; and maximum adsorption of
an IgG antibody onto a polymer has been observed to occur at pH 7.
Thus, in accordance with one or more embodiments, and as disclosed
in an article by Turkmen, et al. entitled "Phenylalanine Containing
Hydrophobic Nanospheres for Antibody Purification" in Biotechnol.
Prog., 24, 2008, pp. 1297-1303, a liquid containing the antibody is
maintained at a pH of about 6-8 during the adsorption process. In
accordance with one or more embodiments that use a polymer
substrate structure, a method for adsorbing antibodies onto such a
polymer substrate structure, as disclosed in an article by Qian et
al. entitled "Immobilization of Antibodies on Ultraflat Polystyrene
Surfaces" in Clinical Chemistry, 46:9, 2000, pp. 1456-1463,
comprises: (a) adding antibodies until they reach a concentration
of 0.001-10 mg/mL to a coating buffer, for example, a 40-60 mmol/L
carbonate buffer at about pH 9.0-10.5; (b) wetting the polymer
substrate structure by immersion, rinsing or spraying with the
antibody-buffer mixture; (c) incubating the wet polymer substrate
structure for about 7-9 hours at about 1-10 degrees Celsius; (d)
rinsing the polymer substrate structure with deionized water to
remove excess antibody-buffer mixture; and (d) drying the polymer
substrate structure with nitrogen. In accordance with one or more
such embodiments, and as disclosed in an article by Boyd et al.
entitled "Application of Antibody Adsorbed Polyester Cloth for
Rapid Screening of Elution Conditions for Antigen
Immunopurification" in Immunological Investigations, Volume 25,
Issue 5&6, September 1996, pp. 447-453, the polymer substrate
structure can be made of, for example and without limitation,
polystyrene or polyester.
[0050] Antibody Adsorption onto Gold: Although, from a bonding
strength standpoint, covalent linking of an antibody to a substrate
structure is stronger than adsorbing the antibody onto a substrate
structure, the inventor has discovered that, in accordance with one
or more embodiments of the present invention, antibodies adsorbed
onto a gold substrate provides bacterial immobilization--for
example, see an article by Suo et al. entitled "Efficient
Immobilization and Patterning of Live Bacterial Cells" in Langmuir
2008, 24, pp. 4161-4167 which describes a method for adsorption of
antibodies on a gold substrate. Advantageously, this can simplify
manufacturing processes by avoiding a need to covalently link
antibodies to a gold substrate to fabricate immobilization
particles.
[0051] Coating Substrate Support with Gold: In accordance with one
or more embodiments of the present invention, a gold substrate is
applied, for example, by deposition onto a substrate support, for
example and without limitation, a polymer substrate support. Prior
to applying the gold substrate, the substrate support may need to
be cleaned. The substrate support may be cleaned with soap and
water, and rinsed with water and/or by suitable cleaning methods
described above. Alternatively, depending upon how clean the
substrate support is after a prior manufacturing step, the
substrate support can be cleaned using a glow discharge. If, as
determined routinely by one of ordinary skill in the art, the
substrate support is too dirty, the gold, or if necessary, a
preceding chrome or titanium substrate layer, might not stick to
the substrate support, thereby reducing the area for attachment of
immobilization molecules. In accordance with one or more further
embodiments, a substrate is comprised of several metal layers
including, for example and without limitation, gold/titanium layers
or gold/titanium/tungsten (for example, 20% titanium, 80% tungsten)
layers, which titanium or titanium/tungsten layers have a thickness
in a range from about 5 to about 100 Angstroms, and which titanium
or titanium/tungsten layers are applied, for example, by
deposition, to the substrate support prior to applying a gold
layer. The titanium can be deposited using a process such as, for
example and without limitation, a vacuum sputtering process, or any
other deposition process capable of depositing such titanium or
titanium/tungsten layers. In accordance with one or more
embodiments, gold can be deposited using a vacuum sputtering
process, and the gold substrate layer may have a thickness in a
range from about 5 to about 200 Angstroms. If the gold substrate
layer is too thin, it might not form a continuous layer, and
instead, it will form islands of gold and voids in gold coverage
that will provide a less effective bonding area for immobilization
molecules. Although the gold substrate layer can be made thicker
than 100 Angstroms, it may not be required to be thicker, and it
will be less expensive to keep the gold substrate layer to a
thickness of about 100 Angstroms.
[0052] In accordance with one or more further embodiments, to help
promote adhesion of immobilization molecules to the substrate
structure, a chromium substrate layer is deposited, for example and
without limitation, vacuum deposited, to a thickness in a range
from about 0.1 to about 5 nm on a substrate support, and this is
followed by applying a gold substrate layer thereon having a
thickness in a range from about 5 to about 100 nm.
[0053] Cleaning the Gold Surface: In accordance with one or more
embodiments, the surface of a gold substrate may be cleaned using,
for example and without limitation, the following method, wetting
the gold substrate surface with a boiling solution of
H.sub.2O.sub.2 (35%), NH.sub.3 (25%) and Milli-Q water in a 1:1:5
ratio mixture for 10 minutes and by rinsing in Milli-Q water, which
method is disclosed in an article by Schmid et al. entitled
"Site-directed antibody immobilization on gold substrate for
surface plasmon resonance sensors" in Sensor and Actuators B:
Chemical, Vol. 113, Issue 1, 17 Jan. 2006, pp. 297-303 (the Schmid
article").
[0054] Maintaining Gold Cleanliness: Once a gold substrate layer is
deposited, its surface should be kept clean enough for successful
adsorption of the immobilization molecules. The surface of the gold
substrate layer can be kept clean using one or more of the
following procedures to handle the substrate after deposition. Such
procedures include, for example and without limitation, High
Efficiency Particulate Absorbing (HEPA) filtration of air coming
into contact with the gold substrate surface; transporting the gold
substrates in a clean container; and ensuring that workers coming
into close contact with the gold substrates wear clean room gowns,
facemasks, eye protection, and so on. If the gold substrate surface
becomes contaminated, it will require cleaning steps such as, for
example and without limitation, one or more of the methods
described above, soap and water, and/or glow discharge cleaning of
the substrate material.
[0055] Covalent Linking Antibody to a Gold Substrate: In accordance
with one or more embodiments, antibodies are covalently linked to a
gold substrate that is attached to a polymer support substrate. A
method for covalently linking antibodies to gold surfaces is
disclosed, for example and without limitation, in an article by
Siiman et al. entitled "Covalently Bound Antibody on Polystyrene
Latex Beads: Formation, Stability, and Use in Analysis of White
Blood Cell Populations" in J. of Colloid and Interface Science,
234, 2001, pp. 44-58, which article is incorporated by reference
herein.
[0056] Covalently Linking an Antibody to a Gold Substrate Using a
Thiol
[0057] In accordance with one or more embodiments, antibodies are
covalently linked to a gold substrate. One method for attaching an
antibody containing a thiol group to gold is to attach a thiol
functional group contained in an antibody to the gold substrate. As
is well known, a thiol functional group is an organic compound that
contains a sulfur and hydrogen group (i.e., --SH). Suitable such
methods for covalently linking antibodies to a gold substrate are
disclosed in the following articles, which articles are
incorporated by reference herein: an article by Karyakin et al.
entitled "Oriented Immobilization of Antibodies onto the Gold
Surfaces via Their Native Thiol Groups", in Anal. Chem., 72(16),
2000, pp. 3805-3811; and the Schmid article.
[0058] Another method for attaching an antibody to a gold substrate
comprises attaching a thiol-containing compound to the gold
substrate, and then linking the antibody to the thiol-containing
compound. A suitable thiol-containing compound is, for example and
without limitation, dithiobis(succinimidyl undecanoate)
("DSU")--when DSU is chemisorbed onto a gold substrate, it induces
amine reactive sites to be formed on the gold surface. In
accordance with one or more such embodiments, a thiol such as, for
example and without limitation, is attached to the substrate in a
manner described below, and an aptamer or an antibody is attached
to the thiol. One such method for covalently linking antibodies to
a gold substrate is disclosed in an article by Mosher, et al.
entitled "Microminiaturized Immunoassays Using Atomic Force
Microscopy and Compositionally Patterned Antigen Arrays" in Anal.
Chem., 70, 1998, pp. 1233-1241 (the "Mosher article") and
comprises, for example and without limitation: (a) wetting top
surfaces 105, 206 or 305 of gold substrates 101, 201 or 302,
respectively, by immersion, rinsing or spraying with a dilute
ethanolic solution of DSU (at a concentration in a range from about
0.1 to about 1.0 mM) for a length of time in a range from about 8
to about 24 hours; (b) adding antibodies (until they reach a
concentration in a range from about 0.5 to about 2.0 mg/mL) to 50
mM of Dulbecco's phosphate buffer (PBS) (Dulbecco's PBS is
available from Life Technologies accessible at
www.lifetechnologies.com) having a pH in a range from about 5.5 to
about 6.5; and (c) wetting gold top surfaces by immersion, rinsing,
or spraying with the antibody-buffer mixture (for example,
immersion in the antibody-buffer mixture for a length of time in a
range from about 30 minutes to about 12 hours, where 90 minutes is
normally sufficient).
[0059] Another method for covalently linking antibodies to a gold
substrate is also disclosed in the Mosher article and comprises:
(a) wetting top surfaces 105, 206 or 305 of gold substrates 101,
201 or 302, respectively, with a solution of dithiobissuccinimide
propionate (DSP) in dimethysulfoxide (DMSO) (at a concentration in
a range from about 0.00005M to about 0.00050M) at room temperature
for a length of time in a range from about 1.5 to about 2.5 hours;
(b) rinsing the substrate with DMSO; (c) rinsing the substrate with
PBS having a pH in a range from about 6.9 to about 7.9 (in
accordance with a method disclosed in the Schmid article); (d)
adding antibodies (until they reach a concentration in a range from
about 0.5 to about 2.0 mg/mL) to 50 mM of Dulbecco's PBS having a
pH in a range from about 5.5 to about 6.5; (e) wetting gold
surfaces by immersion, rinsing or spraying with the antibody-buffer
mixture (for example, by immersion in the antibody-buffer mixture
for a length of time in a range from about 30 minutes to about 12
hours where 90 minutes is normally sufficient).
[0060] Still another method for covalently linking antibodies to a
gold substrate is also disclosed in the Mosher article, which
method yields more antibodies having their Fc fragments attached to
the gold substrate at a side opposite target microorganism or
chemical binding fragments of the antibodies. Fc fragments are
portions of an antibody that do not attach to antigens. The method
comprises: (a) wetting top surfaces 105, 206 or 305 of gold
substrates 101, 201 or 302, respectively, with a solution of DSP in
DMSO (at a concentration in a range from about 0.0002M to about
0.0010M) at room temperature for a length of time in a range from
about 1.5 to about 2.5 hours; (b) rinsing the substrate with DMSO;
(c) rinsing the substrate with PBS having a pH in a range from
about 6.9 to about 7.9; (d) covalently attaching a Protein A layer
to a thiol linked gold substrate by soaking the gold substrate a
length of time in a range from about 4 to about 10 hours at a
temperature in a range from about 1 to about 8 degrees Celsius in a
Protein A solution in a phosphate buffer (the solution having a
concentration in a range from about 0.1 to about 4 mg/ml); (e)
wetting the gold substrate with an ethanolamine hydrochloride
solution having a concentration in a range from about 0.1 to about
3 M and having a pH in a range from about 7.6 to about 9.6 for a
length of time in a range from about 0.1 to about 2 hours to block
residual reacting sites; (f) washing the substrate with distilled
water; (g) drying the substrate in accordance with a method
disclosed in the Schmid article; (h) adding antibodies (until they
reach a concentration in a range from about 0.5 to about 2.0 mg/mL)
to about 40 to about 60 mM of Dulbecco's PBS having a pH in a range
from about 5.5 to about 6.5; (i) wetting gold surfaces by
immersion, rinsing or spraying with the antibody-buffer mixture
(for example, by immersion in the antibody-buffer mixture for a
length of time in a range from about 30 minutes to about 12 hours
where 90 minutes is normally sufficient.
[0061] Covalently Linking an Antibody to a Gold Substrate Using
Biotin and Streptavidin: In accordance with one or more
embodiments, immobilization particles are made by depositing
streptavidin on a gold substrate, attaching biotin to an antibody
and/or antibody fragment and attaching the biotin-conjugated
antibody and/or antibody fragment to the streptavidin, thereby
forming a bond between the biotin and streptavidin. A method for
bonding antibodies to a gold surface using biotin and streptavidin
is disclosed in an article by Kim et. al. entitled "Selective
immobilization of proteins on gold dot arrays and characterization
using chemical force microscopy" in J. of Colloid and Interface
Science, 334, 2009, pp. 161-6, which article is incorporated by
reference herein. In accordance with one or more such embodiments,
octadecanethiol (ODT) may be applied to a gold substrate to prevent
adhesion of antibodies, for example and without limitation, in
areas of the substrate such as near and around edges, to prevent
adhesion of antibodies.
[0062] Immobilization Particle with an Aptamer
[0063] It is believed that aptamers have several advantages over
antibodies in fabricating immobilization particles in accordance
with one or more embodiments of the present invention. For example,
aptamers may be synthesized without using animals or other live
organisms, and therefore, they may have minimal batch-to-batch
variation, which batch-to-batch variation is typical of antibody
manufacture. In addition, aptamers are non-toxic, relatively
non-immunogenic, and they bind to whole cell or molecular targets
with similar affinity and specificity as antibodies. In further
addition, aptamers are small so they may be less susceptible to
steric interference. In still further addition, aptamers can
penetrate into a cell's interior for secure attachment. In yet
still further addition, DNA aptamers are more thermally stable than
antibodies. Lastly, aptamers are readily mass-produced and this
makes them less expensive to use than antibodies.
[0064] Aptamer Selection: Once a target microorganism or molecule
is identified and isolated, it is can be used to select an aptamer
to fabricate one or more embodiments of the present invention. In
accordance with one or more embodiments, aptamers are, for example
and without limitation, one or more of the following: a DNA, an RNA
and a peptide. In accordance with one or more embodiments, aptamers
suitable for use in fabricating one or more embodiments include
aptamers selected in accordance with one or more methods disclosed
in the following articles (which articles are incorporated by
reference herein): (a) an article by Tuerk, et al. entitled
"Systematic evolution of ligands by exponential enrichment: RNA
ligands to bacteriophage T4 DNA polymerase" in Science, 249, 3 Aug.
1990, pp. 505-510; (b) an article by Shamah et al. entitled
"Complex Target SELEX" in Accounts of Chemical Research, Vol. 41.
No 1., January 2008, pp. 130-138; (c) an article by Homann et al.
entitled "Combinatorial selection of high affinity RNA ligands to
live African trypanosomes" in Nucleic Acids Research, Vol. 27, No.
9, January 2008 pp. 2006-2014; (d) an article by Wang et al.
entitled "In vitro selection of novel RNA ligands that bind human
cytomegalovirus and block viral infection" in RNA, 6, 2000, pp.
571-583; (e) an article by Keefe et al. entitled "SELEX with
modified nucleotides" in Current Opinion in Chemical Biology, 12,
2008, pp. 448-456; (f) an article by Hamula et al. entitled
"Selection of Aptamers against Live Bacterial Cells" in Anal.
Chem., 80, 2008, pp. 7812-7819; and (g) an article by Hall et al.
entitled "In Vitro Selection of RNA Aptamers to a Protein Target by
Filter Immobilization" in Current Protocols in Molecular Biology,
October 2009, pp. 24.3.1-24.3.27. As is well known to those of
ordinary skill in the art, the SELEX process, the Complex Target
SELEX process and the Counter SELEX process are selection processes
that use a starting library of oligonucleotides and oligopeptides
(approximately 10.sup.5) that contain randomized regions.
[0065] Aptamer Selection--Autoimmune Mimics: In accordance with one
or more embodiments, aptamers are selected to immobilize one or
more autoimmune mimics. Such autoimmune mimics include, for example
and without limitation: (a) tryptophan peptide from myelin basic
protein (see an article by Westall et al. entitled "Essential
chemical requirements for induction of allergic encephalomyelitis"
in Nature, 229, 1971 pp. 22-24); (b) mid-region from myelin basic
protein (see an article by Shapira et al. entitled "Biological
activity and synthesis of an encephalitogenic determinant" in
Science, 172, 1971, pp. 736-738); (c) hyperacute site from myelin
basic protein (see an article by Westall et al. entitled
"Hyperacute autoimmune encephalomyelitis-unique determinant
conferred by serine in a synthetic autoantigen" in Nature, 269,
1977 pp. 425-427); (d) S-antigen 375-386 (see an article by Dua et
al. entitled "Structure-function studies of s-antigen: use of
proteases to reveal a dominant uveitogenic site" in Autoimmunity,
10, 1991, pp. 153-163); (e) acetylcholine receptor 129-145 (see an
article by Yoshikawa et al. entitled "A 17-mer self-peptide of
acetylcholine receptor binds to B cell MHC class II, activates
helper T cells, and stimulates autoantibody production and
electrophysiologic signs of myasthenia gravis" in J. Immunol. 159,
1997, pp. 1570-1577); (f) acetylcholine receptor 67-75 (see an
article by Bellone et al. entitled "The main region of the
nicotinic acetylcholine receptor" in J. Immunol., 143, 1989, pp.
3568-3579); (g) Sm b/B' protein proline region (see an article by
James et al. entitled "Side-chain specificities and molecular
modeling of peptide determinants for two anti-Sm B/B'
autoantibodies" in J. Autoimmunity, 12, 1999, pp. 43-49); and (h)
tropomyosin isomer V 4-10 (see an article by Vera et al. entitled
"Tropmodulin-binding site mapped to residues 7-14 at the N-terminal
heptad repeats of tropomyosin isoform 5" in Arch. Biochem. Biophys,
378, 2000, pp. 16-24). Suitable aptamers which immobilize the
above-identified mimics are selected using any one of a number of
methods that are well known to those of ordinary skill in the art
routinely and without undue experimentation, for example and
without limitation, using one of the SELEX type of processes.
[0066] Aptamer Selection--Immobilizing Bacteria, Fungi, Viruses,
Serpins and Chemicals: In accordance with one or more embodiments,
aptamers are selected to immobilize, for example and without
limitation, one or more of the following genera of bacteria (use of
such immobilization particles provides a method of treating or
mitigating symptoms of diseases associated with such genera of
bacteria): (a) for Multiple Sclerosis, the following genera of
bacteria, Enterococcus, Streptococcus, Lactobacillus, Bacteroides,
Escherichia, Clostridium, Serratia, Bifidobacterium and
Fusobacterium; (b) for ulcerative colitis, the following genera of
bacteria, Burkholderia, Mycobacterium, Bacillus, Clostridium and
Methylobacterium; (c) for Lupus, the following genera of bacteria,
Burkholderia, Mycobacterium, Pseudomonas, Methylobacterium, Vibrio
and Clostridium; (d) for Uveoretinitis, the following genera of
bacteria, Bacteriodes, Bacillus, Clostridium, Lactobacillus,
Fusobacterium, Vibrio, Ruminococcus and Methylococcus; and (e) for
rheumatoid arthritis, the following genera of bacteria, gram
positive bacteria. It is believed that removing these genera of
bacteria from a host is useful as they can exacerbate symptoms
corresponding to the identified diseases. Suitable aptamers which
immobilize the above-identified genera of bacteria are selected and
manufactured using any one of a number of methods that are well
known to those of ordinary skill in the art routinely and without
undue experimentation, for example and without limitation, using
one of the SELEX type of processes. Suitable aptamers which can
serve as immobilization molecules to immobilize the
above-identified genera of bacteria are readily commercially
available, for example and without limitation, Lactobacillium
Acidophilus (hemag1P) is a suitable aptamer that is available from
Aptagen, LLC. (accessible at http://www.aptagen.com).
[0067] In accordance with one or more embodiments, aptamers are
selected to immobilize, for example and without limitation, one or
more of the following genera of fungi: Saccharomyces and Candida.
Suitable aptamers which immobilize the above-identified genera of
fungi are selected and manufactured using any one of a number of
methods that are well known to those of ordinary skill in the art
routinely and without undue experimentation, for example and
without limitation, using one of the SELEX type of processes.
[0068] In accordance with one or more embodiments, aptamers are
selected to immobilize, for example and without limitation, one or
more of the following viruses: Influenza, Herpes, and
Cytomegalovirus. Suitable aptamers which immobilize the
above-identified viruses are selected and manufactured using any
one of a number of methods that are well known to those of ordinary
skill in the art routinely and without undue experimentation, for
example and without limitation, using one of the SELEX type of
processes. Suitable aptamers which can serve as immobilization
molecules to immobilize the above-identified genera of viruses are
readily commercially available, for example and without limitation,
Human Influenza A virus H3N2 (P30-10-16) is a suitable aptamer that
is available from Aptagen, LLC. (accessible at
http://www.aptagen.com).
[0069] In accordance with one or more embodiments, aptamers are to
immobilize serpins--as is well known to those of ordinary skill in
the art, serpins are a group of proteins that inhibit proteases.
Suitable aptamers which immobilize the serpins are selected using
any one of a number of methods that are well known to those of
ordinary skill in the art routinely and without undue
experimentation, for example and without limitation, using one of
the SELEX type of processes.
[0070] In accordance with one or more embodiments, aptamers can be
used to immobilize chemicals, such as but not limited to, the
chemical theophylline--as is well known to those of ordinary skill
in the art, theophylline is found in tea leaves. Suitable aptamers
which can serve as immobilization molecules to immobilize the
above-identified food chemical are readily commercially available,
for example and without limitation, Anti-theophylline is a suitable
aptamer that is available from GeneLink, Inc. (accessible at
http://www.genelink.com).
[0071] Aptamer Selection--pH Effects: The gastrointestinal tract
has a large range of pH from approximately 1 to 8. In accordance
with one or more embodiments of the present invention, when
performing an aptamer selection process using the SELEX process, or
its variants, a solution containing aptamers undergoing a selection
process should be in the same range of pH that one would expect
immobilization of the target microorganism or chemical to occur.
For example, if a target microorganism were to be found in the
small intestine, one ought to select an aptamer in a solution
having a pH in a range from about 2 to about 6. If the target
microorganism were to be found in the large intestine, one ought to
select an aptamer in a solution having a pH in a range from about 6
to about 8.
[0072] Aptamer Adsorption onto Polymer: In accordance with one or
more embodiments, aptamers are adsorbed onto a polymer substrate in
accordance with suitable methods disclosed in the literature such
as a method disclosed, for example and without limitation, in an
article by Balamurugan et al. entitled "Surface Immobilization
Methods for aptamer diagnostic applications" in Analytical and
Bioanalytical Chemistry, vol. 390, issue no. 4, February 2008,
which article is incorporated by reference herein.
[0073] Aptamer Adsorption onto Gold: In accordance with one or more
embodiments, aptamers are adsorbed onto a gold substrate in
accordance with suitable methods disclosed in the literature such
as a method disclosed, for example and without limitation, in an
article by Wang et al. entitled "Aptamer biosensor for protein
detection using gold nanoparticles" in Analytical Biochemistry,
vol. 373, issue no. 2 Feb. 2008, which article is incorporated by
reference herein.
[0074] Covalently Linking an Aptamer to a Gold Substrate: In
accordance with one or more embodiments, aptamers are covalently
linked onto a gold substrate in accordance with suitable methods
disclosed in the literature such as a method disclosed, for example
and without limitation, a method disclosed in an article by Saran
et al. entitled "Micromechanical Detection of Proteins Using
Aptamer-Based Receptor Molecules" in Anal. Chem., 76, 2004, pp.
3194-3198 disclosing how an 5' etiolated aptamer is immobilized on
gold, which article is incorporated by reference herein.
[0075] Covalently Linking an Aptamer to a Gold Substrate Using
Biotin: In accordance with one or more embodiments, aptamers are
covalently linked onto a gold substrate in accordance with suitable
methods disclosed in the literature such as a method disclosed, for
example and without limitation, a method disclosed in an article by
Liss et al. entitled "An Aptamer-Based Quartz Crystal Protein
Biosensor" in Anal. Chem., 74, 2002, pp. 4488-4495 disclosing how
5' biotinylated aptamer is immobilized on streptavidin fixed on a
gold surface with DSP, which article is incorporated by reference
herein.
[0076] Fabricating Immobilization Particles Having Concavities in a
Polymer Substrate/Polymer Substrate Support or Concavities in a
Gold Substrate/Polymer Substrate Support:
[0077] Polymers are moldable/castable/injettable, and in accordance
with one or more embodiments of the present invention, the
immobilization particle comprises a substrate structure fabricated
as (a) a molded/cast/inkjetted polymer substrate/polymer substrate
support; or (b) a gold substrate/polymer substrate support. In
accordance with one or more such embodiments, a mold is made from a
silicon or ceramic wafer using standard manufacturing methods used
to manufacture semiconductor circuits and MEMS. For example and
without limitation, in accordance one or more such embodiments, a
mold is made from a silicon wafer that is micro-machined using
semiconductor manufacturing equipment (other molding substrates may
be used if non-wafer manufacturing equipment and processes are
used). The method comprises the following: (a) spinning (or vapor
phase depositing) and baking photoresist onto a top, polished
surface of a silicon wafer (for example and without limitation, a
wafer having a diameter in a range from about 4'' to about 12'');
(b) preparing a photo mask having a grid pattern; (c) exposing the
photoresist-coated wafer (or other molding substrate) in a suitable
stepper and developing the photoresist coating to expose the
pattern; (d) dry or wet etching to a depth in a range of about 0.01
microns to about 500 microns (the depth is based on an aspect ratio
of depth:feature width in a range from about 001:1 to about
5:1)--for dry etching, one may use a standard silicon wafer etcher
such as an Applied Materials eMAX chamber, a Producer chamber, or a
HART chamber with a standard dry etching process--which chambers
are available from Applied Materials, Inc. of Santa Clara, Calif.;
and for wet etching, one may use standard semiconductor techniques
for wet etching silicon, quartz or other ceramic material wafers
(if molding substrates other than silicon are used, one can use
appropriate etching techniques that are well known to those of
ordinary skill in the art to create a mold to make immobilization
regions in the polymer substrate as well as patterned regions for
spacers); (e) cleaning and drying the wafer or other molding
substrate; (f) applying a release chemical according to
manufacturer's specification to prevent a castable polymer from
sticking to the etched wafer (any one of a number of suitable
release chemicals are well known to those of ordinary skill in the
art) (in accordance with one or more embodiments of the present
invention, the castable polymer can be, for example and without
limitation, polyurethane); (g) pouring and/or inkjetting the
castable polymer onto the wafer or other molding substrate; (h)
placing a sandwiching silicon wafer onto the cast polymer and
applying an amount of pressure required to ensure uniform thickness
of molded cavities (the amount of pressure can be determined by one
of ordinary skill in the art routinely and without undue
experimentation); (i) curing the castable polymer according to
manufacturer's specification; (j) separating the silicon wafers
from both sides of the cast polymer material; (k) cleaning the cast
polymer to remove the release chemical (for example, the cleaning
step can entail washing with semiconductor grade acetone followed
by semiconductor grade isopropyl alcohol); (l) skip to step (n) if
the substrate is not gold, sputtering gold and other required metal
films as described above onto a side of the cast polymer material
having cavities to a thickness in a range from about 40 to about
500 Angstroms (the deposition may use a sputtering tool that is
well known to those of ordinary skill in the art such as, for
example and without limitation, an Applied Materials Endura PVD
chamber with a gold and/or other metal targets with any one of a
number of sputtering process recipes that are well known to those
of ordinary skill in the art--an Applied Materials Endura PVD
chamber is available from Applied Materials, Inc.); (m) removing
the wafer from the sputtering tool; (n) applying a protective film,
such as, for example and without limitation, a pressure sensitive
adhesive kapton film or a PTFE film onto the flat, cavity-less side
of the cast polymer material; (o) if the substrate is gold,
covalently linking antibodies to the gold substrate deposited on
the cast polymer material substrate support as described above;
(o') if the substrate is not gold, applying antibodies to the
surface of the cast polymer material substrate as described above;
(p) as an optional step to remove antibodies adsorbed outside the
cavities, placing the cast polymer material, cavity-side down, onto
a chemical mechanical planarization (CMP) pad of an Applied
Materials Reflexion CMP machine for a time duration in a range from
about 1 to about 100 seconds, using a platen speed in a range from
about 10 to about 200 RPM, a head rotation speed in a range from
about 10 to about 200 RPM, a head membrane pressure in a range from
about 0.2 to about 20 PSI for each zone, and DI water with no
slurry in an amount in a range from about 0.5 to about 10 liter/min
(an Applied Materials Reflexion CMP machine is available from
Applied Materials, Inc.); (q) removing the protective pressure
sensitive adhesive film from the underside of the cast polymer
material; (r) aligning the cast polymer material into a mounting
support so that an excimer, infra-red or near infra-red laser can
cut each cavity into individual immobilization particles, the laser
power density being set to a range from about 10 to 1,000,000
mW/cm.sup.2 and the linear feed rate being set to a range from
about 0.1 to about 1000 cm/sec; and (s) laser cutting each
immobilization particle and separate the particles. In accordance
with one or more further embodiments, the immobilization particles
can be cut by, for example and without limitation, a sharp cutting
tool, an electrical discharge, thermally, a water saw, blasted
abrasive materials, and any one of a number of other processes that
are well known to those of ordinary skill in the art.
[0078] Immobilization Particles Having Antibodies and/or Aptamers
Adsorbed onto a Porous Polymer Substrate and Polymer Spacers:
[0079] In accordance with one or more embodiments, an
immobilization particle includes a substrate structure comprised of
a substrate/substrate support which is comprised of a woven, porous
polymer such as, for example and without limitation, polyester,
polystyrene, polyurethane, nylon, fabric, paper or filter or a thin
film such as, for example and without limitation, Mylar. In
accordance with one or more such embodiments, the polymer material
may be a sheet of the polymer fabric, paper, filter or thin film.
For example, polyester films, such as Ultra-Polyester, are
available in rolled films that are thicker than 1.5
microns--although the useful thickness is limited by handling
concerns during manufacturing, thinner is better because there is
more surface-to-volume area available. Making immobilization
particles with higher surface-to-volume ratios is advantageous in
that it enables a host, for example and without limitation, a
mammal, to ingest smaller volumes of immobilization particles to
immobilize target microorganism(s) and/or chemical(s). For full
production, the sheet of polymer paper/film used to fabricate a
substrate/substrate support can be on a roll which is manufactured
in a continuous process using suitable processing equipment. In
accordance with one or more embodiments, a method of making an
immobilization particle comprises: (a) cleaning a paper/film/fabric
polymer substrate in accordance with one or more of the methods for
cleaning polymer described above; (b) adsorbing immobilization
molecules (for example and without limitation, an aptamer and/or an
antibody) onto a side of the polymer having concave features, such
as: (i) concave features made using a mold; or (ii) concave
features made by pressure using a die, or a polymer having surfaces
created by voids in between fibers of a fabric; (c) fabricating
spacers to prevent immobilized target microorganisms and/or
chemicals from communicating with a host's tissue; and (d) cutting
the polymer paper/film/fabric/filter substrate/substrate support
(using one or more of the above-identified cutting methods) into
pieces that are small enough to avoid digestive blockage if the
immobilization particles are to be swallowed (if the immobilization
particles are to be used as a dressing, they can be cut into larger
pieces). In particular, a suitable immobilization particle can be
in the form of: (a) a square having a side whose length is in a
range from about 10 microns to about 5000 microns or (b) a circle
having a diameter whose length is in a range from about 10 microns
to about 5000 microns. If an immobilization particle is to be used
as a dressing, the fabric, paper, filter or film polymer substrate
can be cut in suitable larger pieces. Note, that in cutting a
polymer paper/film/fabric/filter substrate/substrate support into
pieces for GI use, the cutting action should destroy immobilization
molecules disposed at, as well as any immobilization molecules
disposed a small distance inbound from, the perimeter of the cuts
to prevent immobilized targets from being disposed too close to an
edge of the particle. In accordance with one or more such
embodiments, the distance of a cut from the perimeter ought to be
at least at large as the largest dimension of an intended
immobilization target. Thus, for example, for a target bacterium
having a largest dimension of 5 microns, immobilizing molecules
need to be destroyed to a distance of about 5 microns from the
perimeter.
[0080] In accordance with one or more embodiments, a method for
fabricating spacers comprises the following steps. First,
inkjetting and/or silkscreening features, for example and without
limitation, dots, on one or both sides of a polymer
paper/film/fabric/filter substrate/substrate support, wherein (a)
the height of the features is in a range from about 1 micron to
about 500 microns, i.e., a height which is taller than a sum of the
longest dimension of the intended target microorganism and/or
chemical and the longest dimension of the immobilization molecule;
(b) the spacing between the features is in a range from about 1
micron to about 500 microns; and (c) a ratio between the diameter
and height of the features is approximately 1:1 to prevent
breakage. It should be understood that the features do not have to
be circular, and in accordance with one or more embodiments, the
features can have any suitable shape, as long as such features are
tall enough and spaced closely enough together to prevent
immobilized target microorganisms or chemicals from interacting
with a host's tissue. In accordance with one or more such
embodiments, the features can be made of the classes/types of
materials mentioned above, such as, for example and without
limitation, polymers. In addition, polymers such as polyurethane
can be added to the substrate/substrate support by inkjetting.
[0081] The method of fabricating spacers further comprises heating
the polymer substrate/substrate support (for example and without
limitation, a polymer paper/film/fabric/filter) beyond its glass
transition temperature to soften the polymer and to enable it to
sag or get pushed into a form (if the substrate/substrate support
sags into a form by gravity, the deformation process will not
require a tool to push the material into the form). To heat the
polymer substrate/support substrate, the material can be heated
before a tool pushes the polymer substrate/support substrate into
the form or a tool can provide the heat locally (for example and
without limitation, upon contact) to the spacer. To keep the heated
area local to the spacer, the heating process can take place in
vacuum to prevent conductive heat transfer through the air. If a
heated tool is used to push the substrate/support substrate, the
heated tool may also be used to inactivate immobilization molecules
by denaturing, hiding, or destroying them on and/or near the
spacers upon contact. The step of inactivating assures that there
is no host response when high points of spacer features come into
contact with intestinal or other bodily tissue. If immobilization
molecules are to be disposed on both sides of a substrate/substrate
support, the high spots need to be pushed in and out, respectively,
of the substrate/substrate support plane (like an egg carton) so
that there are high spots on both sides of the substrate.
[0082] An alternative method of fabricating spacers comprises
impacting the substrate/support substrate with a tool with enough
force to form the substrate/support substrate. In accordance with
one such method, the substrate does not have immobilization
molecules attached prior to impacting. During impacting, sufficient
force is applied to create concave dimples with adjacent raised
high spots or embossments that inhibit a host's response when high
spots of spacer features touch intestinal, or other bodily, tissue.
The amount of force used may be determined by one of ordinary skill
in the art routinely and without undue experimentation. Next,
immobilization molecules are attached using one or more of the
above-identified methods. Next, immobilization molecules are
neutralized (for example and without limitation, inactivation or
removal) at predetermined locations by, for example and without
limitation, ion beam milling. In accordance with another such
method, immobilization molecules are attached prior to impacting.
To simultaneously neutralize (for example and without limitation,
inactivate or remove) immobilization molecules at predetermined
locations and create the spacers, sufficient force is applied to
crush and inactivate immobilization molecules and to create concave
dimples with adjacent raised high spots or embossments. The amount
of force used may be determined by one of ordinary skill in the art
routinely and without undue experimentation.
[0083] Immobilization Particles Having Antibodies and/or Aptamers
Covalently Linked onto a Gold Substrate/Porous Polymer Substrate
Support and Polymer Spacers:
[0084] In accordance with one or more embodiments, an
immobilization particle comprises a gold substrate and substrate
support comprised of a woven, porous polymer, such as, for example
and without limitation, polyester, polystyrene, polyurethane,
nylon, fabric, paper, filter or thin film such as, for example and
without limitation, Mylar. In accordance with one or more such
embodiments, the polymer material may be a sheet of the polymer
fabric, paper, filter or thin film. For example, polyester films,
such as Ultra-Polyester, are available in rolled films that are
thicker than 1.5 microns--although the useful thickness is limited
by handling concerns during manufacturing, thinner is better
because there is more surface-to-volume area available. In
accordance with one or more such embodiments, a method of making
immobilization particles comprises: (a) cleaning a
paper/film/fabric polymer substrate support in accordance with one
or more of the methods for cleaning polymer described above; (b)
sputtering gold and/or any other metal film in accordance with any
one of such methods described above onto the side of the polymer
having concave features, such as, but not limited to, dimples,
blind holes, and spaces between fibers to a thickness in a range
from about 40 to about 500 Angstroms, both sides of the porous
polymer may be coated with metal if needed; (c) covalently linking
antibodies to the gold substrate in accordance with one or more
such methods described above; (d) fabricating polymer spacers to
prevent immobilized microorganisms or chemicals from communicating
with a host's tissues in accordance the methods described above;
and (e) cutting the polymer paper/film/fabric/filter
substrate/substrate support into pieces that are small enough to
avoid digestive blockage if the immobilization particles are to be
swallowed.
[0085] Immobilization Particles Having Antibodies and/or Aptamers
Adsorbed onto Interior Surfaces of a Tube:
[0086] Some types immobilization particles, such as immobilization
particle 700 described above in conjunction with FIG. 10, resemble
a tube and have immobilization molecules attached to their interior
surfaces. In accordance with one or more such embodiments, a
substrate is extruded into a tube. Once the tube is drawn, a fluid
containing immobilization molecules (for example a fluid having one
or more of the above-described chemistries) is introduced into the
hollow space of the tube to permit the immobilization molecules to
become attached to the substrate. After the immobilization
molecules become attached, the tube is cut using one or more of the
above-described methods to appropriate lengths to create
immobilization particles.
[0087] Alternative Method for Removing Immobilization Molecules
Near Edges and Openings of Immobilization Particles:
[0088] As described above, it is desirable to remove immobilization
molecules from regions of a substrate that may come in contact with
a host's tissue. As described above, masking chemicals such as, for
example and without limitation, ODT can be used to prevent adhesion
of immobilization molecules. Alternatively, immobilization
molecules can be selectively removed from a substrate by ion
milling, excimer laser or by polishing.
[0089] Delivery Technology:
[0090] Since one use of immobilization particles fabricated in
accordance with one or more embodiments of the present invention is
to deliver the immobilization particles to a host's
gastrointestinal tract, the immobilization particles can be
delivered orally or via the anus. For oral delivery, a capsule, a
tablet, a particle or a liquid form of delivery can be used.
Depending upon the location of target microorganisms or chemicals,
one can tailor the type of delivery method that is required.
[0091] pH Delivery: If the target delivery region is the large
intestine, and the immobilization particles need to avoid
deployment anywhere above the large intestine, one could
encapsulate the immobilization particles inside a pH triggered
capsule (the stomach's pH is approximately 1-2 (empty) and 3-4
(with food) due to stomach acid, and the pH of the gastrointestinal
tract gradually increases along the small intestine until it
reaches approximately 7 at the entrance of the large intestine). In
accordance with one or more such embodiments, the immobilization
particles are placed inside an open gel-cap, and then the gel-cap
would be closed. Once closed, the gel-cap would be coated with a
coating that dissolves when the pH of the environment reaches
approximately 7. Examples of a suitable pH triggered coating
include, for example and without limitation, pharmaceutical grade
shellac or Eudragit-S polymer. A standard method of coating the
capsules is to place the capsules in a rotating coating pan where
the coating is sprayed onto the capsules while hot dry air is blown
as the capsule tumbles inside the rotating coating pan.
[0092] Coatings: If the target delivery region is the small
intestine, in accordance with one or more embodiments, the
immobilization particles could be placed inside a gel-cap that
would survive stomach acid and would dissolve upon entry into the
small intestine. If the target delivery is a specific location in
the small intestine, then a coating could be used for the gel-cal
where the coating's triggering pH is tuned to open at the desired
location in the small intestine. In accordance with one or more
such embodiments, suitable coatings can be comprised of, for
example and without limitation: (a) a pH sensitive
poly(meth)acrylate copolymer such as, for example and without
limitation, Eudragit FS, Eudragit S(-100), Eudragit RL, Eudragit
RS(-100) or Eudragit L(-100); (b) ethylcellulose; (c) shellac; (d)
deesterified pectin; (e) polygalacturonic acid (PGA or its
potassium and sodium salts); (f) vinyl acetate resin; (g)
carboxylated polyvinyl acetates; (h) polyvinyl/maleic anhydride
copolymers; (i) ethylene/maleic anhydride copolymers; (j)
methylacrylic acid/methyl methacrylate copolymers; (k) waxes; and
(l) chitosan-calcium-alginate, as disclosed in: (i) an article by
Sriamornsak et. al. entitled "Composite Film-Coated Tablets
Intended for Colon-Specific Delivery of 5-Aminosalicylic Acid:
Using Deesterified Pectin" in Pharm Dev and Tech., Vol. 8, No. 3,
2003, pp. 311-318; (ii) an article by Hua et al. entitled
"Technology to Obtain Sustained Release Characteristics of Drugs
after Delivered to the Colon" in J. of Drug Targeting, Vol. 6,
Issue 6, July 1999, pp. 439-448; (iii) an article by Rudolph et al.
entitled "A new 5-ASA multi-unit dosage form for the therapy of
ulcerative colitis" in European J. of Pharmaceutics and
Biopharmaceutics., Vol. 51, Issue 3, May 2001, pp. 183-190; (iv) an
article by Gupta et al. entitled "A novel pH- and time-based
multi-unit potential colonic drug delivery system. I. Development"
in International J. of Pharmaceutics, Vol. 213, Issues 1-2, 1 Feb.
2001, pp. 83-91; and (iv) U.S. Pat. No. 5,401,512 entitled "Delayed
release oral dosage forms for treatment of intestinal
disorders."
[0093] Although many aptamers and antibodies can be stored as
manufactured, some aptamers and antibodies may require an inert
environment to protect them from oxygen or other sources of harm.
In such case, an inert purge gas such as, for example and without
limitation, nitrogen or argon can be used during manufacture of
capsules. Also, a dry mixture of minerals and/or vitamins can be
added to the immobilization particles. Examples of such minerals
are, for example and without limitation, magnesium, selenium,
manganese, iron, chromium, calcium, iodine, chloride, sodium,
potassium, boron, bromide, silicon, phosphorus, titanium, rubidium,
cobalt, copper, antimony, molybdenum, strontium, zinc, nickel,
tungsten, scandium, vanadium, tellurium, tin, lanthanum, yttrium,
silver, gallium, bismuth, zirconium, cerium, cesium, gold,
beryllium, hafnium, samarium, terbium, europium, gadolinium,
dysprosium, thorium, holmium, lutetium, erbium, ytterbium,
neodymium, praseodymium, niobium, tantalum, thallium, rhenium,
indium and so forth. These minerals can be added using a trace
mineral powder complex manufactured by Trace Minerals Research of
Ogden, Utah. The minerals can also be added individually or in
mixtures by powders supplied by many nutritional supplement
ingredient companies.
[0094] Packaging: In accordance with one or more embodiments,
immobilization particles can be packaged into a sachet for storage
and use and later ingested alone or with food. In accordance with
one or more embodiments, immobilization particles can be delivered
in a cap of a drink bottle where a patient breaks a seal and mixes
the particles into the drink prior to consumption. In accordance
with one or more embodiments, immobilization particles can be
premixed in a liquid drink or foodstuff.
[0095] Coated Immobilization Particles: In accordance with one or
more embodiments, an immobilization particle comprises enteric
coatings, applied to individual immobilization particles or applied
to a cluster of immobilization particles. Such immobilization
particles can be ingested in a non-capsule or non-tablet form, and
may be delivered to specific locations in the small intestine or in
the large intestine. In accordance with one or more embodiments,
enteric-coated immobilization particles can be packaged into a
sachet for storage and later consumed alone or with food. In
accordance with one or more embodiments, enteric-coated
immobilization particles can be delivered in a cap of a drink
bottle where a patient breaks a seal and mixes the immobilization
particles into the drink prior to consumption. In accordance with
one or more embodiments, enteric-coated immobilization particles
can be premixed in a liquid drink or foodstuff. The coatings can be
comprised of, for example and without limitation, (a) a pH
sensitive poly(meth)acrylate copolymer such as, for example and
without limitation, Eudragit FS, Eudragit S(-100), Eudragit RL,
Eudragit RS(-100) and Eudragit L(-100); (b) ethylcellulose; (c)
shellac; (d) deesterified pectin; (e) polygalacturonic acid ("PGA")
or its potassium or sodium salts; (f) vinyl acetate resin; (g)
carboxylated polyvinyl acetate; (h) polyvinyl/maleic anhydride
copolymer; (i) ethylene/maleic anhydride copolymer; (j)
methylacrylic acid/methyl methacrylate copolymer; (k) wax; and (l)
chitosan-calcium-alginate, see an article by Sriamornsak et. al.
entitled "Composite Film-Coated Tablets Intended for Colon-Specific
Delivery of 5-Aminosalicylic Acid: Using Deesterified Pectin" in
Pharm Dev and Tech., Vol. 8, No. 3, 2003, pp. 311-318; an article
by Hua et al. entitled "Technology to Obtain Sustained Release
Characteristics of Drugs after Delivered to the Colon" in J. of
Drug Targeting, Vol. 6, Issue 6, July 1999, pp. 439-448; an article
by Rudolph et al. entitled "A new 5-ASA multi-unit dosage form for
the therapy of ulcerative colitis" in European J. of Pharmaceutics
and Biopharmaceutics., Vol. 51, Issue 3, May 2001, pp. 183-190; an
article by Gupta et al. entitled "A novel pH- and time-based
multi-unit potential colonic drug delivery system. I. Development"
in International J. of Pharmaceutics, Vol. 213, Issues 1-2, 1 Feb.
2001, pp. 83-91; and U.S. Pat. No. 5,401,512 entitled "Delayed
release oral dosage forms for treatment of intestinal disorders."
Since a patient can consume one or more embodiments of
immobilization particles, in such form of delivery, a patient may
think of the immobilization particles as a food product rather than
a medical pill. Advantageously, this can have a psychological
benefit that can improve patient compliance. In accordance with one
or more embodiments, a flavored coating may be placed over the
enteric coating to further disguise the medication as a food
product and to further encourage compliance.
[0096] Liquid Delivery: In accordance with one or more embodiments,
immobilization particles can be delivered in a mixture with a
non-toxic liquid such as a food-based oil or water-based saline
solution inside a sealed capsule. Appropriate preservative
chemicals such as, for example and without limitation, antioxidants
can be added to the liquid. Examples of suitable anti-oxidants that
can be added include, for example and without limitation, vitamin
C, vitamin E, alpha-lipoic acid, uric acid, selenium, a carotenoid,
super oxide dismutase, resveratrol and pycnogenol. Medicinal herbs
can also be added to the liquid. Examples of suitable medicinal
herbs that can be added include, for example and without
limitation, aloe vera, Cat's Claw, Echinacea and Golden Seal. If an
oil is used as a liquid, probiotics such as, for example and
without limitation, Lactobacillus can be used in conjunction with
and/or mixed with the immobilization particles.
[0097] Anal Delivery: In accordance with one or more embodiments,
immobilization particles can be delivered via the anus in a
suppository or enema form. The suppository can be a capsule
containing immobilization particles. For an enema, the
immobilization particles would be carried in a liquid. The
immobilization particles and liquid can be stored in a plastic
bottle until the enema need be ready for use. A nozzle on the
plastic bottle would permit comfortable and safe delivery of the
liquid and immobilization particles into the lower large
intestine.
[0098] In accordance with one or more embodiments, various
chemicals can be added to the enema to help treat an illness.
Examples of suitable chemicals that can be added include, for
example and without limitation, butyric acid, bismuth-containing
compounds, alpha-lipoic acid, super oxide dismutase, Vitamin E,
Vitamin C, Cat's Claw and aloe vera.
[0099] Topical Delivery: In accordance with one or more
embodiments, immobilization particles can be applied topically to
control infection or a microbiome on the skin, in the nasal and/or
sinus cavity, in urogenital areas, in the ear and in the vaginal
tract. In accordance with one or more such embodiments, the
immobilization particles can be mixed into a topical cream or gel.
In accordance with one or more further such embodiments, the
immobilization particles can be mixed into an irrigation liquid or
a gas.
[0100] In accordance with one or more embodiments, the
immobilization particles can be embedded into a bandage to cover a
wound or to control a microbiome locally.
[0101] Embodiments described above are exemplary. As such, many
changes and modifications may be made to the description set forth
above by those of ordinary skill in the art while remaining within
the scope of the invention. In addition, materials, methods, and
mechanisms suitable for fabricating embodiments have been described
above by providing specific, non-limiting examples and/or by
relying on the knowledge of one of ordinary skill in the art.
Materials, methods, and mechanisms suitable for fabricating various
embodiments or portions of various embodiments described above have
not been repeated, for sake of brevity, wherever it should be well
understood by those of ordinary skill in the art that the various
embodiments or portions of the various embodiments could be
fabricated utilizing the same or similar previously described
materials, methods or mechanisms. As such, the scope of the
invention should be determined with reference to the appended
claims along with their full scope of equivalents.
* * * * *
References