U.S. patent application number 13/625770 was filed with the patent office on 2013-02-14 for compositions, methods and uses for biosynthetic plasmid integrated capture elements.
The applicant listed for this patent is Maomian FAN, Eric A. HOLWITT, Johnathan L. KIEL, Jill PARKER, Mark SLOAN, Amanda TIJERINA, Melanie WOITASKE. Invention is credited to Maomian FAN, Eric A. HOLWITT, Johnathan L. KIEL, Jill PARKER, Mark SLOAN, Amanda TIJERINA, Melanie WOITASKE.
Application Number | 20130040305 13/625770 |
Document ID | / |
Family ID | 43527403 |
Filed Date | 2013-02-14 |
United States Patent
Application |
20130040305 |
Kind Code |
A1 |
KIEL; Johnathan L. ; et
al. |
February 14, 2013 |
COMPOSITIONS, METHODS AND USES FOR BIOSYNTHETIC PLASMID INTEGRATED
CAPTURE ELEMENTS
Abstract
Embodiments herein report compositions, systems and methods for
making and using plasmid vectors and nanotube complexes. In certain
embodiments, compositions, systems and methods herein include
making plasmid vectors having aptamer inserts. In some embodiments,
methods disclosed herein may be used to rapidly generate large
quantities of plasmid vectors having aptamer inserts directed to a
particular target agent. Other aspects concern plasmid constructs
associated with organic semiconductors. Yet other aspects concern
complexes of nanotubes associated with dsDNA aptamers and tracking
molecules.
Inventors: |
KIEL; Johnathan L.;
(Universal City, TX) ; TIJERINA; Amanda; (San
Antonio, TX) ; HOLWITT; Eric A.; (Brooks City-Base,
TX) ; PARKER; Jill; (Brooks City-Base, TX) ;
SLOAN; Mark; (Spring Branch, TX) ; WOITASKE;
Melanie; (La Vernia, TX) ; FAN; Maomian; (San
Antonio, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KIEL; Johnathan L.
TIJERINA; Amanda
HOLWITT; Eric A.
PARKER; Jill
SLOAN; Mark
WOITASKE; Melanie
FAN; Maomian |
Universal City
San Antonio
Brooks City-Base
Brooks City-Base
Spring Branch
La Vernia
San Antonio |
TX
TX
TX
TX
TX
TX
TX |
US
US
US
US
US
US
US |
|
|
Family ID: |
43527403 |
Appl. No.: |
13/625770 |
Filed: |
September 24, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12792492 |
Jun 2, 2010 |
|
|
|
13625770 |
|
|
|
|
61183453 |
Jun 2, 2009 |
|
|
|
Current U.S.
Class: |
435/6.15 ;
435/320.1; 435/6.1; 435/91.41; 536/23.1; 977/700; 977/742; 977/773;
977/774; 977/810; 977/894; 977/920 |
Current CPC
Class: |
B82Y 5/00 20130101; C12N
15/111 20130101; C12N 2320/32 20130101; C12N 2330/00 20130101; C12P
19/34 20130101 |
Class at
Publication: |
435/6.15 ;
435/320.1; 435/91.41; 536/23.1; 435/6.1; 977/774; 977/810; 977/773;
977/700; 977/894; 977/920; 977/742 |
International
Class: |
C12N 15/63 20060101
C12N015/63; G01N 21/64 20060101 G01N021/64; C12N 15/64 20060101
C12N015/64; C07H 21/04 20060101 C07H021/04 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of Contract No. F41624-D-7000 awarded by the United States Air
Force.
Claims
1-24. (canceled)
25. A plasmid complex composition comprising: a plasmid having one
or more unselected aptamers inserted into the plasmid to make a
random aptamer-plasmid complex, wherein the unselected aptamers are
not yet selected to bind to a target; and an organic semiconductor
associated with the one or more plasmids, wherein the random
aptamer-plasmid complex and the organic semiconductor forms random
aptamer-plasmid-organic semiconductor complex.
26. The composition of claim 25, further comprising the random
aptamer-plasmid-organic semiconductor complex associated with
nanoparticles or microbeads.
27. The composition of claim 26, wherein the nanoparticles
immobilize the random aptamer-plasmid-organic semiconductor
complex.
28. The composition of claim 26, wherein the nanoparticles or
microbeads are selected from the group consisting of paramagnetic
nanoparticles, quantum dots, nanostructures, colloidal gold,
colloidal silver, iron nanoparticles, platinum nanoparticles,
microspheres, or nanospheres.
29. The composition of claim 25, wherein the organic semiconductor
comprises DAT or DALM or other organic semiconductor.
30. The composition of claim 25, wherein the target comprises a
protein, peptide, polysaccharide, lipid, or nucleic acid.
31. The composition of claim 26, wherein the random
aptamer-plasmid-organic semiconductor complex is further
immobilized on a solid substrate.
32. The composition of claim 25, wherein the target comprises a
virus, a yeast, a spore, a bacterium, a disorder-associated
molecule, a disease-progression molecule, a disease marker or a
combination thereof.
33. A method for producing a plasmid complex directed to bind a
target molecule comprising: inserting unselected aptamers into
plasmids having one or more selectable markers to make a
plasmid-random aptamer complex, wherein the unselected aptamers are
not yet selected to bind a target; introducing the plasmids to a
host organism capable of producing organic semiconductors; allowing
the organic semiconductors to associate with the plasmids to form a
plasmid-random aptamer-organic semiconductor complex; obtaining one
or more target molecules associated with nanoparticles to make
target molecule-nanoparticle complexes; introducing target
molecule-nanoparticle complexes to the host organism having
plasmid-random aptamer-organic semiconductor complex constructs or
to extracted plasmid-random aptamer-organic semiconductor complex
constructs; isolating the plasmid-random aptamer-organic
semiconductor complex constructs associated with the target
molecule-nanoparticle complexes; and separating specific
plasmid-random aptamer-organic semiconductor complexes that bind to
the target molecule to generate a selected aptamer plasmid organic
semiconductor complex.
34. The method of claim 33, further comprising: introducing the
selected aptamer plasmid organic semiconductor complexes to a
random aptamer-plasmid construct-free bacterial or mammalian
culture to make selected aptamer-plasmid construct producing
clones; introducing the culture to a selection media wherein only
the cultures taking up the selected aptamer-plasmid organic
semiconductor complexes propogate, and cloning the selected
aptamer-plasmid-organic semiconductor complexes.
35. The method of claim 34, further comprising, growing the clones
on media, wherein the media permits synthesis of organic
semiconductors and wherein the organic semiconductor associates
with the selected aptamer-plasmid-organic semiconductor complexes
making an organic semiconductor selected aptamer-plasmid complex
capable of associating with the target molecule.
36. The method of claim 33, wherein target molecule comprises a
protein, peptide, polysaccharide, lipid, or nucleic acid.
37. The method of claim 33, further comprising immobilizing the
organic semiconductor-specific aptamer-plasmid complex on a
surface.
38. A complex composition comprising: a dsDNA random aptamer
associated with a fluorescent agent and a carbon nanotube to form a
complex comprising a carbon nanotube-random dsDNA
aptamer-fluorescent agent, wherein the dsDNA aptamer binds a target
agent.
39. The composition of claim 38, wherein the complex is associated
with nanoparticles or microbeads.
40. The composition of claim 38, wherein the nanoparticles
immobilize the complex.
41. The composition of claim 38, wherein the nanoparticles or
microbeads are selected from the group consisting of paramagnetic
nanoparticles, quantum dots, nanostructures, colloidal gold,
colloidal silver, iron nanoparticles, platinum nanoparticles,
microspheres, or nanospheres.
42. The composition of claim 38, wherein the fluorescent agent
comprises quantum dots or other fluorescent agent capable of being
quenched by the carbon nanotube.
43. The composition of claim 38, wherein the target agent comprises
a protein, peptide, polysaccharide, lipid, or nucleic acid.
44. The composition of claim 38, wherein the complex is further
immobilized on a solid substrate.
45. The composition of claim 38, wherein the carbon nanotube
further comprises a cell compatible coating.
46. The composition of claim 45, wherein the cell compatible
coating comprises an organic semiconductor.
47. A plasmid-complex composition comprising: a plasmid having one
or more unselected aptamers inserted into the plasmid to make
random aptamer-plasmid complexes, wherein the unselected aptamers
are not yet selected to bind to a target; and an organic
semiconductor, DALM, associated with the one or more plasmids,
wherein the random aptamer-plasmid complexes and DALM form random
aptamer-plasmid-DALM complexes.
48. The composition of claim 47, further comprising the random
aptamer-plasmid-DALM complexes associated with nanoparticles.
49. The composition of claim 48, wherein the nanoparticles
immobilize the random aptamer-plasmid-organic semiconductor
complexes.
50. The composition of claim 47, wherein the nanoparticles comprise
paramagnetic nanoparticles, iron nanoparticles, platinum
nanoparticles or other paramagnetic nanoparticles.
51. The composition of claim 47, wherein the random
aptamer-plasmid-DALM complexes are selected to bind targets.
52. The composition of claim 47, wherein the targets are one or
more proteins or protein complexes.
53. The composition of claim 47, wherein the random
aptamer-plasmid-DALM complexes are further immobilized on a solid
substrate.
54. The composition of claim 47, wherein the targets comprise
bacteria.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit under 35 U.S.C.
119(e) of U.S. Provisional Patent Application Ser. No. 61/183,453
filed on Jun. 2, 2009 which is incorporated herein by reference in
its entirety.
FIELD
[0003] Embodiments herein relate to compositions, systems, and
methods for making and using plasmid vectors. In certain
embodiments, compositions, systems and methods herein include
making plasmid vectors having aptamer inserts. In some embodiments,
methods disclosed herein may be used to rapidly generate large
quantities of plasmid vectors directed to a particular target
agent. In other embodiments, compositions, systems, uses and
methods relate to generating nanotube complexes. Methods, systems
and compositions disclosed herein may be used for detection, drug
development, drug delivery, identification, collection,
decontamination, analysis of disease detection or progression,
neutralization, determination of viability, and/or inactivation or
killing of a target agent.
BACKGROUND
[0004] Aptamers are single-stranded nucleic acids isolated from
random-sequence nucleic acid libraries by selection such as in
vitro selection. Many DNA or RNA sequences have been isolated that
bind a diverse range of targets, including metal ions, small
organic compounds, biological cofactors, metabolites, proteins and
nucleic acids. The target versatility and the high binding affinity
of both DNA and RNA aptamers, their properties of precise molecular
recognition, along with the simplicity of in vitro selection, make
aptamers attractive as molecular receptors and sensing
elements.
[0005] Current methods, techniques and devices used for identifying
chemical and biological analytes typically involve capturing the
analyte through use of a non-specific solid surface or through
capture deoxyribonucleic acids (DNA) or antibodies. A number of
known binding agents must then be applied, particularly in the case
of biological analytes, until a binding agent with a high degree of
affinity for the analyte is identified such as an aptamer. A
labeled aptamer (e.g., labeled DNA) must be applied, where the
aptamer causes, for example, the color or fluorescence of the
analyte to change if the binding agent exhibits affinity for the
analyte (i.e., the binding agent binds with the analyte). The
aptamer may be identified by studying which of the various binding
agents exhibited the greatest degree of affinity for the
analytes.
[0006] Some problems associated with current methods of chemical
and biological agent identification include that a great deal of
time and effort is required to repetitiously generate and apply
each of the known labeled aptamers, until an aptamer exhibiting a
high degree of affinity is found. In addition, once the
identification of a high affinity aptamer is made the synthesis of
multiple copies for use becomes a challenge. Accordingly, these
techniques are not conducive to easy automation. Current methods
are also not sufficiently robust to work in environmental
conditions, for example, heat, dust, humidity or other conditions
that may be encountered, for example, in the field or in a food
processing plant. Portability and ease of use are also problems
seen with current methods for chemical and biological agent
identification.
SUMMARY
[0007] Embodiments herein report compositions, systems and methods
for making and using plasmid vectors. In certain embodiments,
compositions, systems and methods herein include making plasmid
vectors having aptamer inserts. In some embodiments, methods
disclosed herein may be used to rapidly generate large quantities
of plasmid vectors directed to a particular target agent. Methods,
systems and compositions disclosed herein may be used for
detection, drug development, drug delivery, identification,
collection, decontamination, analysis of disease detection or
progression, neutralization, determination of viability, and/or
inactivation or killing of a target agent.
[0008] Some embodiments of the present invention report using
nanotubes as quenching agents in constructs described herein. In
accordance with these embodiments, a nanotube construct may be used
in cellular transfection to deliver a targeting molecule (e.g.
targeting an agent in a eukaryotic cell for destruction, detection
or modification etc.). In certain embodiments, a nanotube construct
may be referred to as a nanobe wherein the nanobe includes, but is
not limited to, a nanoparticle associated with a single-stranded
DNA (ssDNA) consensus sequence capable of hybridizing to a
complementary sequence, the ssDNA associated with a nanotube. In
some embodiments, the nanobe complex can penetrate a cell (e.g. a
eukaryotic cell). In some examples, cells may be transfected in
order to modify cells that then modify an organism (e.g. generate
non-virulent forms, non-pathogenic forms or the like, generate
organisms sensitive to certain types of destruction or to design
self-destructing strains or cells etc.). In other embodiments,
cells may be transfected in order to deliver an agent to the cell
(e.g. a drug, a detection agent, targeting agent etc.).
[0009] In other embodiments, a plasmid complex composition may
include one or more plasmids with at least one selectable marker,
having one or more random aptamers inserted into the plasmid, and
an organic semiconductor associated with the one or more plasmids,
wherein the aptamer inserted plasmids and the organic semiconductor
forms a plasmid-random aptamer-organic semiconductor complex. In
certain compositions, a plasmid-random aptamer-organic
semiconductor complex can further include nanoparticles or
microbeads, wherein the nanoparticles or microbeads non-covalently
associate with the plasmid-random aptamer-organic semiconductor
complex. In other embodiments, plasmid-random aptamer-organic
semiconductor complexes that bind to a target agent through
recognition by random aptamers may be selected before or after
association with nanoparticles or microbeads. Selected plasmid
complexes that bind to the target agent may be cloned by
introduction to a bacterial or mammalian culture or amplified. In
addition, selected plasmid-random aptamer complexes may further be
linked to an organic semiconductor if grown in a bacterial culture
capable of producing organic semiconductors. Alternatively,
selected plasmid-random aptamer complexes may be linked to an
organic semiconductor by synthetic addition of an organic
semiconductor to the selected plasmid-random aptamer complexes.
[0010] In other embodiments, a plasmid complex composition may
include one or more plasmids or dsDNA with at least one selectable
marker, having one or more random aptamers inserted into the
plasmid or linked to the dsDNA and a nanotube (e.g. single-walled
carbon nanotube) associated with the one or more plasmids or dsDNA
and one or more fluor, wherein the aptamer(s) insert and the nanobe
forms a plasmid/dsDNA-random aptamer-fluor-nanobe complex. In
certain compositions, a plasmid/dsDNA-random aptamer-fluor-nanobe
complex can further include nanoparticles or microbeads, wherein
the nanoparticles or microbeads non-covalently associate with the
plasmid/dsDNA-random aptamer-fluor-nanobe complex (or random
aptamer-fluor-nanobe complex). In other embodiments,
plasmid/dsDNA-random aptamer-fluor-nanobe complexes that bind to a
target agent through recognition by random aptamers may be selected
before or after association with nanoparticles or microbeads.
Selected plasmid complexes that bind to the target agent may be
cloned by introduction to a bacterial or mammalian culture. In
addition, selected plasmid-random aptamer complexes may further be
linked to a nanobe if grown in a bacterial culture capable of
producing nanobes. Alternatively, selected plasmid-random aptamer
complexes may be linked to a nanobe by synthetic addition of an
organic semiconductor to the selected plasmid-random aptamer
complexes.
[0011] It is contemplated herein that some constructs (e.g. nanobe
constructs) may be used as a sensor for condition of a cell or cell
component, for example, by detecting the presence or absence of
fluorescence of a construct disclosed herein due to target agent
concentrations, levels, or existence.
[0012] Other embodiments of the present invention may concern using
nanoparticles or microbeads associated with plasmid complexes
disclosed herein to immobilize plasmid complexes or select out
plasmid complexes that specifically bind to a target agent. In
certain embodiments, the nanoparticles or microbeads associate with
the selected plasmid complexes covalently or non-covalently.
Nanoparticles or microbeads can be paramagnetic nanoparticles,
quantum dots, nanostructures, colloidal gold, colloidal silver,
iron nanoparticles, platinum nanoparticles, microspheres, or
nanospheres.
[0013] In some aspects, target agents may include, but are not
limited to, whole organisms, inorganic, organic or biochemical
targets such as a virus, bacteria, yeast, spore, metal ions, small
organic compounds, biological cofactors, metabolites, proteins,
nucleic acids, biological warfare agents, terrorism agents, natural
or genetically modified agents. In other embodiments, a target
agent may be a protein, peptide, antibody, antibody fragment,
polysaccharide, lipid, or nucleic acid.
[0014] Samples contemplated herein can be, but are not limited to,
samples from a subject such as human samples, mammalian samples,
bird samples or reptile samples (e.g. blood, buccal, nasal, tissue,
urine, skin). In some embodiments, a sample can be obtained from a
domesticated animal for example, a dog, cat, bird or farm
animal.
[0015] In addition, samples reported herein can include one or more
samplings from an inanimate object including, but not limited to,
air filters, a solid surface, mail, an outer surface of an object,
a filter, a vent, a duct, an aerosol collected on a filter, an
unknown powder, dusty agent sample, any surface of an object, such
as a counter, wall, a table, a chair, equipment (e.g. military
equipment); or any other surface that a subject may come in contact
with; or a sample from food, soil, or water source.
[0016] In some embodiments, the plasmid complex may be further
immobilized on a solid substrate. Solid substrates contemplated
herein include, but are not limited to, microbeads, magnetic beads,
a microarray, a microtiter plate or other solid surface known in
the art.
[0017] Other aspects of the present invention include methods for
producing a plasmid complex directed to bind one or more target
agents. In accordance with these aspects, methods can include
inserting random nucleic acid aptamers into plasmids having
selectable markers then, making random aptamer-plasmid constructs
in a host organism; obtaining one or more target agents associated
with nanoparticles to make target agent-nanoparticle complexes;
introducing target agent-nanoparticle complexes to the host
organism having the random aptamer-plasmid constructs or to
extracted random aptamer-plasmid constructs; and isolating the
random aptamer-plasmid constructs associated with the target
agent-nanoparticle complexes. In addition, other embodiments for
producing plasmid complexes directed to bind a target agent may
include, but are not limited to, introducing the isolated random
aptamer-plasmid constructs associated with the target
agent-nanoparticle to a random aptamer-plasmid construct-free
bacterial culture to make random aptamer-plasmid construct
producing clones; selecting the clones having the selectable
marker, wherein the clones having the selectable marker have random
aptamer-plasmid constructs associated with the target
agent-nanoparticle complexes and wherein the random aptamer-plasmid
specifically recognizes the target agent. Alternatively, selected
clones may be grown on media, wherein the media permits synthesis
of organic semiconductors by the clone and wherein the organic
semiconductor associates with the selected random aptamer-plasmid
constructs making a organic semiconductor-selected random
aptamer-plasmid complex capable of targeting the target agent.
Organic semiconductor-selected random aptamer-plasmid complex may
be further immobilized on a surface.
[0018] Other embodiments can include methods for using a plasmid
complex disclosed herein. For example, methods are disclosed for
using an organic semiconductor-selected aptamer-plasmid complex.
Other embodiments include methods for using a plasmid/dsDNA random
aptamer-fluor-nanotube complex. Nanotubes and target DNA (e.g
aptamers) can combine to form a stable biosensor or biodetector
complex. Methods may include, but are not limited to, obtaining a
complex; exposing a sample suspected of having a target agent to
the complex; and allowing the complex to bind to the target agent
if present in the sample. Some samples may include, but are not
limited to, inanimate, non-living samples for example, samples from
a solid surface, mail, an outer surface of an object, a filter, a
vent, a duct, field samples, military gear, military equipment,
soil or other inanimate object suspected of having a target agent.
In certain embodiments of the present invention, a complex may be
used to detect, identify or destroy a target agent(s). In
accordance with these embodiments, methods may further include,
exposing the complex bound to a target agent to an energy source
capable of destroying, killing or neutralizing the target
agent.
[0019] Energy sources contemplated herein may include, but are not
limited to, microwave radiation, ultraviolet radiation (UV),
visible light, laser, electron beam radiation, pulsed corona
discharge (non-thermal plasma discharge), other forms of ionizing
radiation, and thermal radiation. Other embodiments may include,
introducing an attractant (e.g. magnetic beads, column
chromatography with streptavidin-biotin or other molecule) to the
complex bound to the target agent and concentrating the complex
bound to the target agent for further analysis using the attractant
to concentrate the target agent. Energy sources contemplated of use
herein may be used for detection, modification or destruction of an
organism or cell or other target agent. Certain embodiments may
include immobilizing a complex contemplated herein on a surface for
example, a solid surface. Solid surfaces for immobilizing a complex
contemplated may include, but are not limited to, glass, plastic,
silicon-coated substrate, macromolecule-coated substrate,
particles, beads, microparticles, microbeads, dipstick, magnetic
beads, paramagnetic beads and a combination thereof.
[0020] Other aspects contemplated herein may include a system for
detecting a complex disclosed herein. Certain embodiments for a
system may include an element for inputting random aptamers in a
reaction vessel; and a component for inputting the random aptamers
into a plasmid having at least one selectable marker of a first
bacterial or first mammalian organism to make random
aptamer-plasmid complexes; an element for isolating the random
aptamer-plasmid complexes and introducing the random
aptamer-plasmid complexes to a second bacterial organism capable of
making organic semiconductors in another reaction vessel wherein
the organic semiconductor associates with the random
aptamer-plasmid complexes; and isolating the organic
semiconductor-selected random aptamer-plasmid complex. Other
embodiments may include, a system having component for selecting
organic semiconductor-selected aptamer-plasmid complexes that bind
to a target agent.
[0021] Other embodiments may include a kit for making and/or using
complexes including, but not limited to, a source of plasmids; a
source of nanoparticles; and a source for generating organic
semiconductors or other quenching agent complexes such as nanobes.
A kit may further include magnetic beads or other material capable
of attracting and/or concentration nanoparticles.
[0022] Example kits may include, but are not limited to, one or
more complexes capable of binding one or more target molecule able
to detect, identify, decontaminate, analyze disease progression,
neutralize, determine viability, inactivate, kill or combination
thereof. In some embodiments, a kit may be generated for both
detecting and destroying, detecting and decontaminating, detecting
and identifying, detecting and neutralizing, detecting and
obliterating, detecting and further analyzing one or more target
agents. Certain kits are directed to particular target molecules
and the target molecules can include, but are not limited to, whole
organisms. In some embodiments, a kit can be used to generate
plasmids having aptamers specific for binding a target molecule or
a kit may already have a lyophilized composition of plasmids having
selected aptamers capable of recognizing a target agent. Some kits
may contain a partially or completely dehydrated composition
including, but not limited to, a bacterial host having a plasmid
harboring a random aptamer library or pre-selected aptamer, the
host bacterial having the capability of producing organic
semiconductors. Alternatively, a kit may include a partially or
completely dehydrated composition of a plasmid harboring a random
aptamer library or pre-selected aptamer capable of being introduced
to a bacteria culture for expansion.
[0023] In certain embodiments, a kit can contain partially or
completely dehydrated complexes reported herein and these complexes
can be re-hydrated and expanded and used to assess the presence
and/or level of a target agent(s). In accordance with these
embodiments, a target agent may be in a sample from a subject
suspected of having or at risk for developing a disorder where the
target agent is indicative of the onset, progression or existence
of the disorder. In addition, a target agent may be present in a
remote region where detection and destruction of the agent may be
required. In accordance with these embodiments, complexes may be
used to select one or more aptamers capable of binding a target
agent, amplifying the selected complex and using the amplified
selected complexes to bind to the target agent and destroy the
agent by introducing a source of energy of the present
invention.
[0024] In some aspects of the invention, beads or particles
contemplated herein can include, but not limited to, paramagnetic
beads, magnetic beads, superparamagnetic beads, streptavidin coated
beads, Reverse Phase magnetic beads, carboxy terminated beads,
hydrazine terminated beads, Silica (sodium silica) beads and IDA
(iminodiacetic acid) modified beads, aldehyde modified beads, Epoxy
activated beads, DADPA-modified beads (beads with primary amine
surface group), biodegradable polymeric beads, amino-polystyrene
particles, carboxyl-polystyrene particles, Epoxy-polystyrene
particles; dimethylamino-polystyrene particles, hydroxy-polystyrene
particles, colored particles, flow cytometry particles, and
sulfonate-polystyrene particles. In accordance with these
embodiments, a target agent may be covalently or non-covalently
linked to a bead or particle. Alternatively, complexes may be
immobilized using any bead or particle reported herein.
[0025] In certain embodiments, the nucleic acid sequences or
aptamers may be sequences of 1 to 1000, 10 to 500, 10 to 250, 10 to
150, 10 to 75, 20 to 60, 15 to 45, 20 to 40 nucleotides or base
pairs in length, a single length, a combination of lengths or
mixture thereof or combination thereof.
[0026] In another embodiment, amplification of selected aptamers
(e.g. from a plasmid complex) that bind a target agent can be used
to generate multiple copies of the aptamers (e.g. DNA), hybrid
molecules or RNA aptamers that bind a target agent. Methods useful
for amplifying the partitioned sequences may include, but are not
limited to, polymerase chain reaction (PCR), the ligase chain
reaction (LCR) Q beta Replicase, an isothermal amplification
method, Strand Displacement Amplification (SDA), Repair Chain
Reaction (RCR), transcription-based amplification systems (TAS),
including nucleic acid sequence based amplification (NASBA) and
3SR.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The following drawings form part of the present
specification and are included to further demonstrate certain
embodiments. The embodiments may be better understood by reference
to one or more of these drawings in combination with the detailed
description of specific embodiments presented herein.
[0028] FIG. 1 represents an exemplary schematic of nucleic acid
nanoparticle and organic semiconductor interactions.
[0029] FIG. 2 represents an exemplary schematic of germ line
transfer and gene transfer in a cell.
[0030] FIG. 3 represent an exemplary schematic of organic
semiconductor-selected aptamer-plasmid complex from a random
aptamer library.
[0031] FIG. 4 represents an exemplary electrophoresis gel used for
isolating nucleic acids after exposure to a plasmid complex.
[0032] FIG. 5 represents an exemplary schematic of aptamer
selection and replication/expansion of predetermined target agent
binding aptamers.
[0033] FIG. 6 represents an exemplary electrophoresis gel
illustrating presence or absence of a predetermined insert (as
shown by asterisk (*)), standards were included in the gel.
[0034] FIG. 7 represents an exemplary electrophoresis gel
processing illustrating presence of a predetermined insert (as
shown by asterisk (*)) after several transformations of a parent
cell line in the presence of organic semiconductor associated
nanoparticles, standards were included in the gel.
[0035] FIG. 8A-8D represent exemplary photographs of views through
an inverted microscope of uptake of various nanoparticles by
exemplary cells.
[0036] FIGS. 9A-9B represent exemplary electrophoresis gels of (A)
positive control cell cultures having an exemplary plasmid complex
and cells exposed to a nanoparticle composition and (B) a different
exposure of (A), human HeLa cells and mouse MHS cells.
[0037] FIG. 10 represents an exemplary graph depicting a comparison
of cells exposed to control compositions, control compositions plus
predetermined selective agent compositions and control compositions
plus predetermined selective agent compositions after a 24 hour
pretreatment with organic semiconductor-coated nanoparticles of
some embodiments of the present invention.
[0038] FIG. 11 represents the fluorescence of fluorophore (or
Q-Dots) quenched by SWCNT (single-walled carbon nanotube).
[0039] FIG. 12 represents fluorescent change after bacterial spores
introduced into a quenching system.
[0040] FIG. 13 represents a gel separation of various
constructs.
[0041] FIG. 14 represents microscopic images illustrating
fluorescence of various constructs.
[0042] FIG. 15 represents microscopic images illustrating
fluorescence after interactions of various constructs.
[0043] FIG. 16 represents microscope images (left side)
illustrating fluorescence after interaction of a cell line with a
construct.
[0044] FIG. 17 represents an electrophoretic gel of constructs
under various conditions of digestion with DNase.
[0045] FIG. 18 represents TEM images indicate of various complexes
that can penetrate the cell membrane.
[0046] FIG. 19 represents TEM images indicate of various complexes
that can penetrate the cell membrane with arrows indicating
intracellular aggregate nanobe structures.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
[0047] As used herein, "a" or "an" may mean one or more than one of
an item.
[0048] "Nucleic acid" can mean DNA, RNA, single-stranded,
double-stranded, hybrid molecules such as RNA/DNA or triple
stranded and any chemical modifications thereof. Virtually any
modification of the nucleic acid is contemplated herein. "Nucleic
acid" encompasses, but is not limited to, oligonucleotides and
polynucleotides. "Oligonucleotide" refers to at least one molecule
of between about 3 and about 100 nucleotides in length.
"Polynucleotide" refers to at least one molecule of greater than
about 100 nucleotides in length. These terms generally refer to at
least one single-stranded molecule, but in certain embodiments also
encompass at least one additional strand that is partially,
substantially or fully complementary in sequence. Thus, a nucleic
acid may encompass at least one double-stranded molecule or at
least one triple-stranded molecule that comprises one or more
complementary strand(s) or "complement(s)." As used herein, a
single stranded nucleic acid may be denoted by the prefix "ss", a
double stranded nucleic acid by the prefix "ds", and a triple
stranded nucleic acid by the prefix "ts."
[0049] Within the practice disclosed herein, a "nucleic acid" may
be of almost any length, from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275,
300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,
3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000
or even more bases in length. In some embodiments, nucleic acid
sequences may be around 10 to around 200 bases in length. In other
embodiments, double-stranded DNA molecules (dsDNA) may be around 2
to 1000, 10 to 500, 10 to 250, 10 to 150, 10 to 75, 20 to 60, or
15-45 base pairs in length. The term "nucleic acid" as used herein
can generally refer to at least one molecule or strand of DNA, RNA
or a derivative or mimic thereof, comprising at least one
nucleobase. A "nucleobase" refers to a heterocyclic base, for
example, a purine or pyrimidine base naturally found in DNA (e.g.
adenine "A," guanine "G," thymine "T" and cytosine "C") or RNA
(e.g. A, G, uracil "U" and C), as well as their derivatives and
mimics. A "derivative" refers to a chemically modified or altered
form of a naturally occurring molecule, while "mimic" and "analog"
refer to a molecule that may or may not structurally resemble a
naturally occurring molecule, but that function similarly to the
naturally occurring molecule. One function of a nucleobase is to
hydrogen bond to other nucleobases. Nucleobases can form one or
more hydrogen bonds ("anneal" or "hybridize") with at least one
naturally occurring nucleobase in manner that may substitute for
naturally occurring nucleobase pairing (e.g. the hydrogen bonding
between A and T, G and C, and A and U).
[0050] A nucleic acid may include, or be composed entirely of, at
least one nucleobase, a nucleobase linker moiety and/or a backbone
moiety.
[0051] As used herein, a "moiety" generally can refer to a smaller
chemical or molecular component of a larger chemical or molecular
structure, and is encompassed by the term "molecule." In some
embodiments, a moiety can be a component of a larger molecule, for
example, a reporter agent moiety or a signal reducing agent
moiety.
[0052] "Aptamer," "DNA capture element" (DCE), "RNA capture
element," or hybrid capture element as used herein can mean
non-naturally occurring nucleic acid molecules (such as nucleic
acid sequences) having a desirable action on a target agent (e.g.
binds to a target agent). In some embodiments, actions on a target
agent can include, but is not limited to, binding, reacting with
covalently attaching target agent; facilitating reaction(s) between
the target agent and another molecule (e.g destruction by an
organic semiconductor exposed to an energy source), killing and/or
neutralizing the target agent. Capture elements or aptamers herein
can include, but are not limited to, nucleic acids that are
generated and/or identified by methods and compositions disclosed
herein. Binding interactions of DCEs or aptamers may not encompass
standard nucleic acid/nucleic acid hydrogen bond formation
exemplified by Watson-Crick basepair formation (e.g., A binds to U
or T and G binds to C), but may encompasses all other types of
non-covalent (or in some cases covalent) binding. Non-limiting
examples of non-covalent binding include hydrogen bond formation,
electrostatic interaction, Van der Waals interaction and
hydrophobic interaction. A DCE or any other aptamer contemplated
herein may bind to another molecule by any or all of these types of
interaction, or in some cases by covalent interaction. Covalent
binding of a DCE to another molecule may occur where the DCE or
target molecule contains a chemically reactive or photoreactive
moiety. The term DCE can include a DNA capture element that is
capable of forming a complex with an intended target agent.
"Target-specific" means that the DCE binds to a target agent with a
much higher degree of affinity than it binds to other
materials.
[0053] "Analyte," "target," "target agent" and "target analyte" as
used herein can mean any compound, whole organism, object, or
aggregate of interest. Target agents can include, but are not
limited to, a whole organism (e.g. bacteria, yeast or virus),
protein, peptide, carbohydrate, polysaccharide, glycoprotein,
lipid, hormone, receptor, antigen, allergen, antibody, substrate,
metabolite, cofactor, enzyme, metal ion, inhibitor, drug,
pharmaceutical, nutrient, toxin, poison, explosive, pesticide,
chemical warfare agent, biohazardous agent, prion, radioisotope,
vitamin, heterocyclic aromatic compound, carcinogen, mutagen,
narcotic, amphetamine, barbiturate, hallucinogen, waste product,
contaminant or other molecule. Molecules of any size can serve as
target agents. "Target agents" are not limited to single molecules,
but may also comprise complex aggregates of molecules, such as a
virus, bacterium, spore, mold, yeast, algae, amoebae,
dinoflagellate, unicellular organism, pathogen or cell. In certain
embodiments, a sample suspected of having a particular bacterium
present, such a pathogenic bacteria, may be a target agent of some
embodiments of the present invention. Virtually any chemical or
biological effector would be a suitable target.
[0054] "Binding" as used herein can mean an interaction,
association or binding between a target agent and an aptamer,
resulting in a sufficiently stable complex so as to permit
separation of aptamer:target complexes from uncomplexed aptamers
under given binding or reaction conditions. Binding is mediated
through hydrogen bonding or other molecular forces.
[0055] "Magnetic bead," "paramagnetic bead," "nanoparticle,"
"magnetic particle" and "magnetically responsive particle" as used
herein can mean any particle dispersible or suspendable in aqueous
media, without significant gravitational settling and separable
from suspension by application of a magnetic field.
[0056] "Bound," as used herein can mean covalently or
non-covalently associated with a molecule.
[0057] "Nanobe," as used herein can mean a nanotube complex or a
complete complex of a single or double-walled carbon nanotube, with
a paramagnetic or other metallic nanoparticle for directing by
magnetic field and enhancing delivery, by electric field focusing,
of functional DNA, RNA or modified nucleic acids into target cells,
and with polymeric coating to facilitate such delivery. Nanobes are
infectious, viral-like synthetic particles that not only can alter
function of the cell but can also propagate the nucleic acid by
incorporation into cellular DNA and by expressing proteins that can
synthesize more DNA or RNA and polymeric coating to deliver that
DNA or RNA to other uninfected cells.
[0058] "Nanotube" as used herein can mean a carbon nanotube
molecule of pure carbon that are long and thin and shaped like
tubes, about 1-3 nanometers in diameter, and hundreds of nanometers
to micrometers long.
[0059] In the following sections, several embodiments of, for
example, compositions and methods are described in order to
thoroughly detail various embodiments herein. It will be obvious to
one skilled in the art that practicing the various embodiments does
not require the employment of all or even some of the specific
details outlined herein, but rather that concentrations, times and
other specific details may be modified through routine
experimentation. In some cases, well known methods or components
have not been included in the description to prevent unnecessary
masking of various embodiments.
[0060] In some embodiments, bacillus spores, botox spores, anthrax
spores, Shiga toxin (protein), Botox toxins (different types), Ms-2
bacterial, Ovalbumin, (protein) Yersinia pestis (vaccine),
Rickettsia (antigen), VEE (Venezuelan Equine Encephalitis),
Vaccinia viruses or a combination of organisms and organism
byproducts are contemplated as target agents for aptamers
contemplated.
[0061] In other embodiments, an aptamer can be selected to bind
small organic molecules that include, but are not limited to,
small, organic ligands, amino acids, nucleotides and derivatives,
biological cofactors, antibiotics or other small molecules from
random aptamer intergrated plasmids. In accordance with these
embodiments, small molecules can include, but are not limited to,
one or more of the following, organic dyes, Theophyllin, lEHT,
Dopamine, Hoechst 33258, sulforhodamine B, Sulforhodamine B,
Cellobiose, D-tryptophan, L-arginine, L-citrullin, L-argininamide,
L-arginine, L-arginine Mirror-image, L-valine, L-isoleucine, AMP,
AMP Mirror-image, Guanosine, FMN, NAD, Vitamin B12, 8-oxo-dG,
5'-cap, Xanthene, Kanamycin A, Lividomycin, Tobramycin, Neomycin B,
Viomycin, Chloramphenicol, Streptomycin, Rev peptide, Vasopressin
Mirror-image and a combination thereof. In certain aspects,
compositions, methods and systems disclosed herein could be
employed in the identification of and/or destruction of target
agents; diagnosis and treatment of disease in a sample; to develop
new compounds for pharmaceutical use, medical or industrial
purposes; or to identify chemical and biological warfare target
agents on a military facility, in an area suspected of harboring
target agents or out in the field.
[0062] Aptamers contemplated herein may be generated by any means
known in the art and used as tools in a variety of ways. Random
aptamers, partially selected pools of aptamers or selected aptamers
that bind to a target agent can be inserted into a plasmid
construct. Random libraries may contain a variety of lengths of
aptamer sequences and insertions into plasmid can be of the same or
different lengths depending on conditions for insertion and sizes
of inserts. For example, methods and compositions described in U.S.
application Ser. No. 11/965,039 filed Dec. 27, 2007 entitled
"Methods and Compositions for Processes of Rapid Selection and
Production of Nucleic Acid Aptamers," incorporated herein by
reference in its entirety, or using Selex or other methods known in
the art may be used to generate a random library of aptamers or
other a specific aptamer for insertion into a plasmid.
[0063] Other aspects concern immobilizing a target molecule on a
suitable surface, such as microbeads or an array, such as a
microarray format. One advantage for using a microarray format is
the ability to examine thousands of molecules in a sample,
simultaneously. In accordance with these aspects, a target molecule
may be immobilized and a plasmid having an aptamer library may be
used to select specific binding molecules against the target
molecule. Some methods used to immobilize a target molecule are
based on covalent, affinity or electrostatic interactions between a
target molecule and a suitable surface. Aptamers that bind to an
immobilized target molecule can be rapidly identified, isolated and
amplified for use in methods and systems disclosed herein.
Uses
[0064] In some embodiments, aptamers may be generated for use
against any target agent contemplated herein. It is contemplated
that aptamers or aptamer-plasmid complexes or
aptamer-fluor-nanotube complexes may be produced for diagnostic
arrays, portable and/or handheld diagnostic/detection devices,
cellular transfection methods, portable kits, or as agents to
control, revert, or eliminate the target agent.
[0065] Some advantages herein include advantages over some current
technologies. One advantage of compositions, methods and systems
disclosed herein include rare binding sequences (e.g. for
generating low frequency aptamers) in the early stages of selection
are preserved (versus eliminated by other technologies) and can be
amplified as a selected clone to a predetermined target agent. In
addition, methods and systems disclosed herein can allow for the
selection and amplification of all binding sequences from the very
first round of selection of a specific clone. This can eliminate
the need for subsequent rounds of selection while providing a
source of one or more specific aptamers permitting a more stable
supply of a select aptamer as a clone. Once an aptamer clone, for
example, can be identified, isolated, and optionally amplified, and
sequenced, these aptamer clones may be subjected to a more rigorous
selection process under more stringent conditions of binding. In
accordance with these embodiments, aptamer clones with a higher
affinity, avidity and greatest specificity can be separately or
simultaneously selected. In certain embodiments, a plasmid-aptamer
complex identified as a clone may be stored or amplified after
storage (e.g. aliquoted and frozen). In certain aspects, a
plasmid-aptamer complex may be associated with an organic
semiconductor or other agent (e.g. nanobe) and the complex stored
in a host organism for later use (e.g. aliquoted, frozen or freeze
dried).
[0066] In other embodiments, using a modified existing flow
cytometer, particle counter or sorter, one could use a hand held
magnetic aptamer or plasmid aptamer complex reader in order to
capture an aptamer or plasmid-aptamer complex associated with
target agents. A captured magnetic complex may be removed from a
capture cassette of the reader and amplified, for example, by
clonal expansion.
[0067] In certain embodiments contemplated herein, luminescent
colloidal semiconductor nanocrystals (e.g. quantum dots, qdot) that
are inorganic fluorophores can be used in methods disclosed herein
to circumvent some of the functional limitations encountered by
organic dyes. In other embodiments, organic dyes known in the art
may be used, if appropriate. Quantum dots are semiconductors whose
conducting characteristics are closely related to the size and
shape of the individual crystal. In general, the smaller the size
of the crystal, the larger the band gap, the greater the difference
in energy between the highest valence band and the lowest
conduction band becomes, therefore more energy is needed to excite
the dot, and concurrently, more energy is released when the crystal
returns to its resting state. One advantage in using quantum dots
is that because of the high level of control possible over the size
of the crystals produced, it is possible to have very tight control
over conductive properties of the material.
[0068] As a consequence or selection benefit, quantum dots of the
same material, but with different sizes, can emit light of
different colors. Quantum dots have distinct optical applications
due to their theoretically high quantum yield. In some embodiments,
quantum dots of different sizes may be used to select different
aptamers or aptamer-plasmid complexes or for aptamer-qdot-nanotube
complexes contemplated of use in methods herein. It is appreciated
that the selection of quantum dot of use in any of the compositions
or methods herein can be optimized depending on the requirements of
a particular use.
[0069] In other embodiments, a plasmid complex composition may
include one or more plasmids with at least one selectable marker
and having one or more random aptamers inserted into the plasmid.
In other embodiments, a plasmid complex may further include an
organic semiconductor associated with the one or more plasmids,
wherein the aptamer inserted plasmids and the organic semiconductor
forms a plasmid-aptamer-organic semiconductor complex. In other
embodiments, plasmid complexes that bind to a target agent through
recognition by random aptamers may be selected. Selected plasmid
complexes that bind to a target agent may be cloned by introduction
to a bacterial or mammalian culture. In addition, aptamers selected
from plasmids containing a random aptamer library to bind a target
agent may be further associated with an organic semiconductor if
grown in a bacterial culture capable of producing organic
semiconductors.
[0070] Other embodiments for producing plasmid complexes directed
to bind a target agent may include, introducing the isolated random
aptamer-plasmid constructs associated with the target
agent-nanoparticle to a random aptamer-plasmid construct-free
bacterial culture, having organic semiconductor synthesis
capabilities, to make random aptamer-plasmid construct producing
clones; selecting the clones having the selectable marker, wherein
the clones having the selectable marker have random aptamer-plasmid
constructs associated with the target agent-nanoparticle complexes
wherein the random aptamer-plasmid specifically recognizes the
target agent. Alternatively, selected clones may be grown on media,
wherein the media permits synthesis of organic semiconductors by
the clone and wherein the organic semiconductor associates with the
selected random aptamer-plasmid constructs making a organic
semiconductor-selected random aptamer-plasmid complex capable of
targeting the target agent. Organic semiconductor-selected random
aptamer-plasmid complex may be further immobilized on a
surface.
Single--Walled Nanotubes
[0071] In some embodiments, nanotubes may be used to reversibly
quench fluorescent agents of some embodiments or complexes
disclosed herein. In accordance with these embodiments, nanotubes
can be used to quench organic chemical fluors and for example,
quantum dots. In addition, carbon nanotubes can be used to directly
penetrate cells and deliver nucleic acid molecules that can for
example, be recovered by amplification (e.g. PCR). In addition,
carbon nanotubes can be used to enhance penetration (e.g. to
deliver an agent or compound to the cell or cell nucleus) or
destruction of a target cell by for example, radiofrequency to
microwave radiation based upon the intensity of the peak E field of
the radiation.
[0072] Some embodiments of the present invention report using
nanotubes as transient quenching agents of fluorescent compounds,
for example, for transfection of constructs or agents into cells
described herein. In accordance with these embodiments, a nanotube
construct may be used in cellular transfection to deliver a
targeting molecule (e.g. targeting an agent in a eukaryotic cell
for destruction, detection or modification etc.). In certain
embodiments, a nanotube construct may be referred to as a nanobe
wherein the nanobe includes, but is not limited to a nanoparticle
associated with a single-stranded DNA (ssDNA) consensus sequence
capable of hybridizing to a complementary sequence, the ssDNA can
be associated with a nanotube. In some embodiments, the nanobe
complex can penetrate a cell (e.g a eukaryotic cell). In some
examples, cells may be transfected in order to modify cells that
then in turn modify an organism (e.g. generate non-virulent forms,
non-pathogenic forms or the like, generate organisms sensitive to
certain types of destruction or to design self-destructing strains
or cells etc.).
[0073] In other embodiments, a plasmid complex composition
disclosed herein may include one or more plasmids or dsDNA with at
least one selectable marker, having one or more random aptamers
inserted into the plasmid or linked to the dsDNA and a nanotube
(e.g. carbon nanaotube) associated with the one or more plasmids or
dsDNA, wherein the aptamer(s) insert and the nanobe forms a
plasmid/dsDNA-random aptamer-fluor-nanobe complex. In certain
compositions, a plasmid/dsDNA-random aptamer-fluor-nanobe complex
can further include nanoparticles or microbeads, wherein the
nanoparticles or microbeads non-covalently associate with the
plasmid/dsDNA-random aptamer-fluor-nanobe complex. In other
embodiments, plasmid/dsDNA-random aptamer-nanobe complexes that
bind to a target agent through recognition by random aptamers may
be selected before or after association with nanoparticles or
microbeads. Selected complexes that bind to a target agent may be
cloned by introduction to a bacterial or mammalian culture. In
addition, selected plasmid-random aptamer complexes may further be
linked to a nanobe if grown in a bacterial culture capable of
producing nanobes. Alternatively, selected plasmid-random aptamer
complexes may be linked to a nanobe by synthetic addition of an
organic semiconductor to the selected plasmid-random aptamer
complexes. In certain embodiments, a complex may be selected that
binds to one or more target agents (e.g. a family of target agents
or consensus sequences of target agents).
[0074] Some embodiments concern carbon nanotube/nanoparticle nanobe
(e.g. metallic) where the nanobe is capable of self-assembly. In
accordance with these embodiments, an organic semiconductor (e.g.
DAT, DALM) can attach to a nanoparticle (e.g. metal) and to DNA
(e.g. dsDNA) which, in turn, associates with a carbon nanotube via
complementary DNA conjugated covalently to the carbon nanotube.
Complexes having a carbon nanotube attached can be transfected into
cells where DNA can code for a predetermined protein etc. For
example, DNA can code for a nitrate reductase component that acts
like an enzyme to synthesize an organic semiconductor (e.g. DALM)
in the host cell. This organic semiconductor can interact with DNA
or RNA produced by the host cell and can in turn attach to an added
metallic nanoparticle (for example, iron that is paramagnetic) and
could again attach to an added carbon nanotube, coated with DNA, by
complementation. The organic semiconductor/DNA or RNA cell product,
by using a disclosed process, can transfer the expressible DNA or
RNA to a naive (or control cell not having the DNA or RNA) host
cell via the carbon nanotube associated with it. In addition, a
supplementary metallic nanoparticle (e.g. iron) and the carbon
nanotube associated with the complex add the capacity to collect
the product by a magnet and to add further electromagnetic field
interaction (microwave E-field focusing) to the properties of the
biosynthetic nanobe for example, for more efficient cellular
transfection or, for killing of a predetermined target. In certain
embodiments, transfection of a cell by complexes disclosed herein
may be tracked or identified based on fluorescence when a complex
interacts with a target molecule and the complex is no longer
quenched.
[0075] In some embodiments, complexes described herein can be used
to kill organisms without the need of additional activating agents.
In accordance with these embodiments, a complex can include, but is
not limited to components of an organic semiconductor-metallic
agent-nanotube, wherein the organic semiconductor can coat the
nanotube intracellularly to modulate kill of a target organism or
cell. In these examples, water can be used as an activator for
production and destruction of an organism, eliminating or reducing
the need for activating agent(s). An energy source disclosed herein
may be used to target the organic semiconductor-coated nanotube for
destruction of an organism or cell(s).
[0076] Other embodiments of the present invention may concern using
nanoparticles or microbeads associated with the selected complexes
to immobilize them. Alternatively, target agents may be immobilized
on a solid surface and used for selecting aptamer-plasmid complexes
that recognize and associate with the target agent. In certain
embodiments, the nanoparticles or microbeads associate with the
selected complexes covalently or non-covalently. Nanoparticles or
microbeads can be, but are not limited to, paramagnetic
nanoparticles, quantum dots, nanostructures, colloidal gold,
colloidal silver, iron nanoparticles, platinum nanoparticles,
microspheres, or nanospheres.
[0077] In certain embodiments, target agents may include, but are
not limited to, whole organisms such as a virus, bacteria, yeast,
spore, metal ions, small organic compounds, biological cofactors,
metabolites, proteins, nucleic acids, biological warfare agents,
terrorism agents, natural or genetically modified agents. In other
embodiments, a target agent may be a protein, peptide, antibody,
antibody fragment, polysaccharide, lipid, or nucleic acid.
[0078] Samples contemplated herein can be, but are not limited to,
samples from a subject such as human samples, mammalian samples,
bird samples or reptile samples (e.g. blood, buccal, nasal, tissue,
urine, skin). In some embodiments, a sample can be obtained from a
domesticated animal for example, a dog, cat, bird or farm animal.
It is contemplated herein that a sample may be obtained from a
subject having or suspected of developing a disorder and the sample
may be screened for the presence or modulation of a target molecule
in order for a health professional to assess the subject's
condition. Disorders may include, but are not limited to, an
infection by bacteria, fungi, virus or the like, or a disorder
(e.g. cancer, heart disease, kidney disease, diabetes) or condition
(e.g. heart condition, intestinal condition).
[0079] In addition, samples reported herein can include one or more
samplings from an inanimate object including, but not limited to,
air filters, ducts, any surface of an object, such as a counter,
wall, a table, a chair, equipment (e.g. military equipment); or any
other surface that a subject may come in contact with; or a sample
from a food, soil or water source.
[0080] Other embodiments may include methods for making and/or
using an organic semiconductor-selected aptamer-plasmid complex or
nanotube-fluor-dsDNA random aptamer complex capable of binding a
target agent. For example, methods may include, but are not limited
to obtaining a complex; exposing a sample suspected of having the
target agent to the complex; and allowing the complex to bind to
the target agent if present in the sample. In certain embodiments
of the present invention, a complex may be used to detect, identify
or destroy a target agent(s). In accordance with these embodiments,
methods may further include, exposing the complex bound to a target
agent to an energy source capable of destroying, killing or
neutralizing the target agent. Energy sources contemplated herein
may include, but are not limited to, microwave radiation,
ultraviolet radiation (UV), visible light, laser, electron beam
radiation, pulsed corona discharge (non-thermal plasma discharge),
other forms of ionizing radiation, and thermal radiation. Other
embodiments may include, introducing an attractant to the complex
bound to the target agent and concentrating the complex bound to
the target agent for further analysis using the attractant to
concentrate the target agent.
[0081] Other aspects contemplated herein may include a system for
detecting an organic semiconductor-selected aptamer-plasmid complex
against a target agent. Certain embodiments for a system may
include an element for inputting random aptamers in a reaction
vessel; and a component for inputting the random aptamers into a
plasmid having at least one selectable marker of a first bacterial
or first mammalian organism to make random aptamer-plasmid
complexes; an element for isolating the random aptamer-plasmid
complexes and introducing the random aptamer-plasmid complexes to a
second bacterial organism capable of making organic semiconductors
in another reaction vessel wherein the organic semiconductor
associates with the random aptamer-plasmid complexes; and isolating
the organic semiconductor-random aptamer-plasmid complex. Systems
for generating random dsDNA aptamer-fluor-nanobes are
contemplated.
[0082] Other embodiments may include, a system having an aspect for
selecting organic semiconductor-random aptamer-plasmid complexes
that bind to a target agent. In one aspect, a system using a kit
having a lyophilized selectable bacterial culture harboring random
aptamer plasmid may be grown up in a media and combined with one or
more immobilized target agent(s) then specific aptamer plasmids are
selected out by the system using an attractant to immobilized
target agent (e.g. magnetic beads). Selectable aptamer-plasmids
that recognize the target agent(s) can be isolated and introduced
to a naive culture (e.g. not having a selectable marker) for
cloning and isolation by portable means, for example in a remote
area. Produced selected aptamer-plasmids that bind and recognize
the target agent(s) can be used for any purpose disclosed
herein.
[0083] Other embodiments may include a kit for making complexes
disclosed herein including, but not limited to, a source of fluors,
a source of plasmids having at least one selectable marker; a
source of random aptamers; and a source for generating organic
semiconductors. Other kits contemplated herein may include a source
of nanotubes (e.g. SWCNT). A kit may further include magnetic beads
or other material capable of attracting and/or concentration
nanoparticles. These kits are capable of providing portability for
generating a specific molecule of a plasmid-selected
aptamer-organic semiconductor complex directed against a target
agent of use in a remote or unsophisticated area, for example, in
the field.
[0084] Example kits may include, but are not limited to, one or
more organic semiconductor-selected random aptamer-plasmid
complexes capable of binding one or more target molecule able to
detect, identify, decontaminate, analyze disease progression,
neutralize, determine viability, inactivate, kill or combination
thereof. Some kits may contain a partially or completely dehydrated
composition including, but not limited to, a bacterial host having
a plasmid harboring a random aptamer library or pre-selected
aptamer, the host bacterial having the capability of producing
organic semiconductors. Alternatively, a kit may include a
partially or completely dehydrated composition of a plasmid
harboring a random aptamer library or pre-selected aptamer pool
capable of being introduced to a bacteria culture for expansion and
use. In addition, other kits may include a hydrated or dehydrated
composition(s) of random or selected aptamer-fluor-nanotubes
complexes for use in embodiments disclosed herein.
[0085] In some aspects of the invention, beads or particles
contemplated herein can include, but not limited to, paramagnetic
beads, magnetic beads, superparamagnetic beads, streptavidin coated
beads, Reverse Phase magnetic beads, carboxy terminated beads,
hydrazine terminated beads, Silica (sodium silica) beads and IDA
(iminodiacetic acid) modified beads, aldehyde modified beads, Epoxy
activated beads, DADPA-modified beads (beads with primary amine
surface group), biodegradable polymeric beads, amino-polystyrene
particles, carboxyl-polystyrene particles, Epoxy-polystyrene
particles; dimethylamino-polystyrene particles, hydroxy-polystyrene
particles, colored particles, flow cytometry particles, and
sulfonate-polystyrene particles. In accordance with these
embodiments, a target agent may be covalently or non-covalently
linked to a bead or particle. Alternatively, complexes may be
immobilized using any bead or particle reported herein.
[0086] In certain embodiments, the nucleic acid sequences or
aptamers may be sequences of 1 to 1000, 10 to 500, 10 to 250, 10 to
150, 10 to 75, 20 to 60, 15 to 45, 20 to 40 nucleotides or
basepairs in length, a single length, a combination of lengths or
mixture thereof or combination thereof. In some aspects, a random
aptamer library can contain random length aptamers or aptamers of
the same or similar lengths may be selected.
[0087] In another embodiment, amplification of aptamers (e.g. from
a plasmid complex or a nanobe complex) that bind a target agent can
be used to generate multiple copies of the aptamers (e.g. DNA),
hybrid molecules or RNA aptamers (e.g. RNA aptamers) that bind a
target agent. Methods useful for amplifying the partitioned
sequences may include, but are not limited to, polymerase chain
reaction (PCR.TM.), the ligase chain reaction (LCR) Qbeta
Replicase, an isothermal amplification method, Strand Displacement
Amplification (SDA), Repair Chain Reaction (RCR),
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR.
[0088] In another embodiment, plasmid complexes may be generated to
a target agent and used to analyze the presence of the target agent
in a sample suspected of containing the target agent. In accordance
with this exemplary use, aptamers directed to bind the target agent
can be combined with the sample and the sample can be analyzed for
the presence, absence or level of the target agent based on
association with the aptamers.
Nucleic Acids
[0089] Nucleic acids within the scope may be made by any technique
known to one of ordinary skill in the art. Examples of nucleic
acids, particularly synthetic oligonucleotides, can include a
nucleic acid made by in vitro chemical synthesis using
phosphotriester, phosphite or phosphoramidite chemistry and solid
phase techniques via deoxynucleoside H-phosphonate intermediates.
In certain embodiments, aptamers contemplated herein can be
generated and may be modified. Examples of modified aptamers
include those that can be modified after amplification reactions
such as PCR..TM. or the synthesis of oligonucleotides. Examples of
a biologically produced nucleic acids include recombinant nucleic
acid production in living cells, such as recombinant DNA vector
production in bacteria.
[0090] Nucleobase, nucleoside and nucleotide mimics or derivatives
are well known in the art, and have been described. Purine and
pyrimidine nucleobases encompass naturally occurring purines and
pyrimidines and derivatives and mimics thereof. These include, but
are not limited to, purines and pyrimidines substituted with one or
more alkyl, carboxyalkyl, amino, hydroxyl, halogen (i.e. fluoro,
chloro, bromo, or iodo), thiol, or alkylthiol groups. The alkyl
substituents may comprise from about 1, 2, 3, 4, or 5, to about 6
carbon atoms.
[0091] In addition, purine and pyrimidine derivatives or mimics can
be used as base substitutions in any of the methods disclosed
herein
Amplification
[0092] In certain embodiments, plasmid-aptamer complexes may be
amplified to provide a source of high affinity nucleic acids for
associating analytes. Amplification may also be of use in the
iterative process for generating arrays with greater specificity or
binding affinity for a target agent. Within the scope,
amplification may be accomplished by any means known in the
art.
Primers
[0093] The term primer, as defined herein, is meant to encompass
any nucleic acid that is capable of priming the synthesis of a
nascent nucleic acid in a template-dependent process. Typically,
primers are oligonucleotides around 5-100 base pairs in length, but
longer sequences may be employed. Primers may be provided in
double-stranded or single-stranded form.
[0094] In some embodiments, amplification of a random region is
produced by mixing equimolar amounts of each nitrogenous base
(A,C,G, and T) at each position to create a large number of
permutations (e.g. where "n" is the oligo chain length) in a very
short segment. This provides dramatically more possibilities to
find high affinity nucleic acid sequences when compared to the
10.sup.9 to 10.sup.11 variants of murine antibodies produced by a
single mouse.
[0095] A number of template dependent processes are available to
amplify the marker sequences present in a given template
sample.
[0096] In other embodiments, other methods for amplification of
nucleic acids, include but are not limited to, the ligase chain
reaction ("LCR"), Qbeta Replicase, isothermal amplification
methods, and Strand Displacement Amplification (SDA) as well as
other methods known in the art. Still other amplification methods
may be used in accordance with embodiments disclosed herein. Other
nucleic acid amplification procedures may include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA). In some of the disclosed
methods, the nucleic acid sequences may be prepared for
amplification by standard phenol/chloroform extraction, heat
denaturation of a clinical sample, treatment with lysis buffer and
mini-spin columns for isolation of DNA and RNA or guanidinium
chloride extraction of RNA. These amplification techniques involve
annealing a primer which has APTAMER specific sequences. Following
polymerization, DNA/RNA hybrids are digested with RNase H while
double stranded DNA molecules are heat denatured again. In either
case the single stranded-DNA is made fully double stranded by
addition of second APTAMER specific primer, followed by
polymerization. The double-stranded DNA molecules are then multiply
transcribed by a polymerase such as T7 or SP6. In an isothermal
cyclic reaction, the RNA's are reverse transcribed into double
stranded DNA, and transcribed once against with a polymerase such
as T7 or SP6. The resulting products, whether truncated or
complete, indicate APTAMER specific sequences.
[0097] Polymerases and Reverse Transcriptases include but are not
limited to thermostable DNA
Polymerases: 0 nmiBase.TM.. Sequencing Enzyme Pfu DNA Polymerase
Taq DNA Polymerase Taq DNA Polymerase, Sequencing Grade
TaqBead.TM.. Hot Start Polymerase AmpliTaq Gold Tfl DNA Polymerase
Tli DNA Polymerase Tth DNA Polymerase DNA POLYMERASES: DNA
Polymerase I, Klenow Fragment, Exonuclease Minus DNA Polymerase
1DNA Polymerase I Large (Klenow) Fragment Terminal Deoxynucleotidyl
Transferase T4 DNA Polymerase Reverse Transcriptases: AMV Reverse
Transcriptase M-MLV Reverse Transcriptase
[0098] For certain embodiments, it may be desirable to incorporate
a label into the nucleic acid sequences such as the aptamer,
amplification products, probes or primers. A number of different
labels can be used, including but not limited to fluorophores,
chromophores, radio-isotopes, enzymatic tags, antibodies,
chemiluminescent, electroluminescent, and affinity labels.
[0099] Examples of affinity labels contemplated herein, can
include, but are not limited to, an antibody, an antibody fragment,
a receptor protein, a hormone, biotin, DNP, and any
polypeptide/protein molecule that binds to an affinity label.
[0100] Examples of enzymatic tags include, but are not limited to,
urease, alkaline phosphatase or peroxidase. Colorimetric indicator
substrates can be employed with such enzymes to provide a detection
means visible to the human eye or spectrophotometrically
visible.
[0101] The following fluorophores disclosed herein can include, but
are not limited to, Alexa 350, Alexa 430, AMCA, BODIPY 630/650,
BODIPY 650/665, BODIPY-FL, BODIPY--R6G, BODIPY-TMR, BODIPY-TRX,
Cascade Blue, Cy2, Cy3, Cy5,6-FAM, Fluorescein, HEX, 6-JOE, Oregon
Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,
Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET,
Tetramethylrhodamine, and Texas Red.
Solid Phase
[0102] It is contemplated herein that any solid phase support may
be used for isolation or immobilization of a separated target agent
or aptamer-plasmid complex of any of the procedures disclosed
herein. In some embodiments, a solid phase component can include
beads or a bead technology for example beads, microbeads,
particles, microparticles, nanoparticles or combination thereof. In
accordance with these embodiments, the beads or particles may be
selected from the group consisting of paramagnetic beads, magnetic
beads, superparamagnetic beads, streptavidin coated beads, Reverse
Phase magnetic beads, carboxy terminated beads, hydrazine
terminated beads, Silica (sodium silica) beads and IDA
(iminodiacetic acid) modified beads, aldehyde modified beads, Epoxy
activated beads, DADPA-modified beads (beads with primary amine
surface group), amino-polystyrene particles, carboxyl-polystyrene
particles, Epoxy-polystyrene particles, dimethylamino-polystyrene
particles, hydroxy-polystyrene particles, colored particles, flow
cytometry particles, sulfonate-polystyrene particles or combination
thereof
Methods of Immobilization of Amplified Aptamers or Target
Molecules
[0103] In various embodiments, amplified population of
aptamer-plasmid complexes may be attached to a solid surface
("immobilized"). In other embodiments, target molecules associated
with nanoparticles may be associated with a solid surface. In one
embodiment, immobilization may occur by attachment of an organic
semiconductor to a solid surface, such as a magnetic bead, a
plastic microtiter plate or a glass slide or a chip material. In
one example, use of a semiconductor for this system is advantageous
in that the attachment of aptamers may be readily reversed by
addition of a chelator, such as EDTA if the attachment is through
magnesium or some other chelatable compound.
[0104] Immobilization of aptamers may alternatively be achieved by
a variety of methods involving either non-covalent or covalent
interactions between the immobilized aptamers, for example, an
anchorable moiety, and an anchor. In some embodiments,
immobilization may be achieved by coating a solid surface with
streptavidin or avidin and the subsequent attachment of a
biotinylated polynucleotide. Immobilization may also occur by
coating a polystyrene or glass solid surface with poly-L-Lys or
poly L-Lys, Phe, followed by covalent attachment of either amino-
or sulfhydryl-modified polynucleotides, using bifunctional
crosslinking reagents by methods known in the art.
[0105] Other solid surfaces contemplated of use may include, but
are not limited to, glass, plastic, silicon-coated substrate,
macromolecule-coated substrate, particles, beads, microparticles,
microbeads, dipstick, magnetic beads, paramagnetic beads and a
combination thereof. In certain embodiments, these solid surfaces
can be used to immobilize an amplified aptamer for further use such
as detecting an analyte or agent in a sample.
[0106] Immobilization may take place by direct covalent attachment
of short, 5'-phosphorylated primers to chemically modified
polystyrene plates. The covalent bond between the modified
oligonucleotide and the solid phase surface is formed by
condensation with a water-soluble carbodiimide. This method
facilitates a predominantly 5'-attachment of the oligonucleotides
via their 5'-phosphates. In addition, attachment to a solid surface
may be made through non-covalently immobilizing aptamer molecules
in the presence of a salt or cationic detergent on a hydrophilic
polystyrene solid support containing an--OH, --C.dbd.O or --COOH
hydrophilic group or on a glass solid support. The support is
contacted with a solution having a pH of about 6 to about 8
containing the aptamer and the cationic detergent or salt. The
support containing the immobilized aptamer may be washed with an
aqueous solution containing a non-ionic detergent without removing
the attached molecules.
[0107] One commercially available method for immobilization is the
"Reacti-Bind..TM.. DNA Coating Solutions" (see
Instructions--Reacti-Bind..TM. DNA Coating Solution). This product
comprises a solution that is mixed with DNA and applied to surfaces
such as polystyrene or polypropylene. After overnight incubation,
the solution is removed, the surface washed with buffer and dried,
after which it is ready for hybridization. It is envisioned that
similar products, i.e. Costar "DNA-BIND.TM." or Immobilon-AV
Affinity Membrane (IAV, Millipore, Bedford, Mass.) may be used in
the practice of the embodiments disclosed herein.
Separation and Quantitation Methods
[0108] In some embodiments, it may be desirable to separate
aptamers of a clone of different lengths for the purpose of
quantitation, analysis or purification. In other embodiments it may
be desirable to separate aptamers of a clone and group them by size
in order to use them for methods disclosed herein.
Gel Electrophoresis
[0109] In one embodiment, aptamers of an aptamer-plasmid clone
(e.g. target agent directed) can be sized by for example, excising
the aptamer of interest and using agarose, agarose-acrylamide or
polyacrylamide gel electrophoresis or other methods known in the
art for sizing an insert.
[0110] Separation by electrophoresis is based upon methods known in
the art. Samples separated in this manner may be visualized by
staining and quantitated, in relative terms, using densitometers
which continuously monitor the photometric density of the resulting
stain. The electrolyte may be continuous (a single buffer) or
discontinuous, where a sample is stacked by means of a buffer
discontinuity, before it enters the running gel/running buffer.
Chromatographic Techniques
[0111] Alternatively, chromatographic techniques may be employed to
effect separation. There are many kinds of chromatography which may
be used for example: adsorption, partition, ion-exchange and
molecular sieve, and many specialized techniques for using them
including column, paper, thin-layer and gas chromatography.
Aptamers Combinational Uses
[0112] Random aptamers may be prepared by any method known in the
art. In addition, aptamers or aptamer-plasmid clones may be used
alone or in combination with other aptamers or other
aptamer-plasmid clones specific for the same or different target
agents. Further, aptamers may specifically include "secondary
aptamers" where a consensus sequence is derived from comparing two
or more isolated aptamers or aptamer-plasmid clones that bind to a
given target.
[0113] In general, a minimum of approximately 3 nucleotides,
preferably at least 5 nucleotides, can effect specific binding. The
binding specificity of the target/agent capture element complexes
disclosed herein concern sufficient sequence to be distinctive in
the binding aptamers and sufficient binding capacity of target
agent(s) to obtain the necessary interaction. Oligonucleotides of
sequences shorter than 10 bases can be feasible if interaction can
be obtained in the context of the environment in which the target
is placed.
[0114] Any aptamer or aptamer-plasmid contemplated herein can
contain a sequence that confers binding specificity, but may be
extended with flanking regions and otherwise derivatized. In one
particular embodiment, aptamer binding sites can be flanked by
known, amplifiable sequences, facilitating the amplification of the
aptamers by PCR or other amplification techniques. In a further
embodiment, flanking sequence(s) may include a specific sequence
that recognizes or binds a moiety to enhance the immobilization of
an aptamer to a substrate.
[0115] Aptamers of interest may include modified oligomers. Any of
the hydroxyl groups ordinarily present in aptamers may be replaced
by phosphonate groups, phosphate groups, protected by a standard
protecting group, or activated to prepare additional linkages to
other nucleotides, or may be conjugated to solid supports. The 5'
terminal OH is conventionally free but may be phosphorylated.
Hydroxyl group substituents at the 3' terminus may also be
phosphorylated. The hydroxyls may be derivatized by standard
protecting groups. One or more phosphodiester linkages may be
replaced by alternative linking groups. These alternative linking
groups include, exemplary embodiments wherein P(O)O is replaced by
P(O)S, P(O)NR.sub.2, P(O)R, P(O)OR', CO, or CNR.sub.2, wherein R is
H or alkyl (1-20 C) and R' is alkyl (1-20 C); in addition, this
group may be attached to adjacent nucleotides through O or S. Not
all linkages in an oligomer need to be identical.
[0116] Aptamers generated or used as starting materials in a
process may be single-stranded or double-stranded DNA. In some
embodiments, sequences are double-stranded DNA. In some
embodiments, aptamers will contain a randomized sequence portion,
generally including from about 10 to 400 nucleotides, more
preferably 20 to 100 nucleotides. Randomized sequences can be
flanked by primer sequences that permit the amplification of
aptamers found to bind to a target agent. Flanking sequences may
also contain other features, such as restriction sites. These
primer hybridization regions can contain for example 10 to 80, or
15 to 40, or 20 to 40, bases of known sequence. Randomized portions
and primer hybridization regions of the initial oligomer population
are preferably constructed using conventional solid phase
techniques. Such techniques are well known in the art. Aptamers may
also be synthesized using solution phase methods such as triester
synthesis, known in the art. For synthesis of some randomized
regions, mixtures of nucleotides at positions where randomization
are desired are added during synthesis.
[0117] Any degree of randomization may be employed. Some positions
may be randomized by mixtures of only two or three bases rather
than the conventional four. Randomized positions may alternate with
those which have been specified. Indeed, it is helpful if some
portions of the candidate randomized sequence are in fact
known.
[0118] In some embodiments, substrates may be used for
immobilization of bacterial cultures harboring plasmids disclosed
herein. Substrates for immobilization may include, but are not
limited to, nitrocellulose, nylon membrane or glass. Numerous other
matrix materials may be used, including, but not limited to,
reinforced nitrocellulose membrane, activated quartz, activated
glass, polyvinylidene difluoride (PVDF) membrane, polystyrene
substrates, polyacrylamide-based substrate, other polymers such as
poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl
siloxane) and photopolymers which contain photoreactive species
such as nitrenes, carbenes and ketyl radicals capable of forming
covalent links with target molecules.
[0119] It is contemplated herein that any of the aptamers used in
compositions or methods can be one specific aptamer or a mixture of
aptamers directed to one or more target agent.
EXAMPLES
[0120] The following examples are included to demonstrate some
embodiments. It should be appreciated by those of skill in the art
that the techniques disclosed in the examples which follow
represent techniques discovered by the inventors to function well
in the practice of embodiments disclosed herein, and thus can be
considered to constitute exemplary modes for its practice. However,
those of skill in the art should, in light of some embodiments,
appreciate that many changes can be made in certain embodiments
which are disclosed and still obtain a like or similar result
without departing from the spirit and scope herein.
Example 1
[0121] BioSPLICE is the acronym for Biosynthetic plasmid integrated
capture element. It represents the culmination of basic and applied
research to solve the problem of generating a biological system
that can detect, identify and neutralize target biological agents
in a seamless manner. The final BioSPLICE system is self-contained,
self-replicating and capable of performing the aforementioned
functions without extensive reagents or instrumentation. The system
has a number of options for reporting and neutralizing the target
biological agent that can incorporate sophisticated external
instrumentation (e.g. Charles River Device, PNNL Green Box, GE DVD
Device, GE RFID concept device, microtiter plate reader for visible
light absorption or fluorescence, or a luminometer for
thermochemiluminescence for "reading" reporters; and cold plasma,
pulsed microwave devices or UV A or visible light irradiatiors for
neutralization) or employ the simplest physical and chemical means
(color or color intensity change, visible to the eye, when bound to
a target agent or activation of bound reporter with hydrogen
peroxide and bicarbonate for killing with heat or sunlight). This
system is versatile and flexible. BioSPLICE can be used to select
new targeting elements in the field and retain this new signature
for further replication and utilization and still provide
biologically encoded information that can be sent reported.
Example 2
[0122] In one exemplary method, compositions (e.g. BioSPL10E
methodologies) involve generating and using recombinant plasmid
vectors containing a DNA aptamer insertion(s) (DNA Capture Element)
in the host bacterium species/strain (e.g. Escherichia coli JM109
or HB101) or a mammalian cell. This plasmid can be extracted and
bound specifically to a molecular target (e.g., protein,
polysaccharide, lipid, or nucleic acid) where the target molecule
is immobilized covalently or non-covalently on a paramagnetic
metallic nanoparticle. The binding can occur prior to or after
plasmid extraction because such nanoparticles can penetrate living
microbes or cells. If random collections of aptamers are present as
plasmid inserts, the one that binds best to the molecular target
linked to a nanoparticle will be selected and isolated. After
magnetic separation of the nanoparticles of the better or best
binding aptamers, they are added to a parent E. coli that does not
contain the plasmid and is, in certain cases, not resistant to
ampicillin, another antibiotic or selectable marker to which the
plasmid conveys resistance.
[0123] After sufficient incubation time for the bacteria (e.g. E.
coli) to take up the plasmid-coated nanoparticles (uncoated
nanoparticles will also be taken up), the bacteria are plated on
solid media (agar) or on a media impregnated filter containing
ampicillin alone or LB agar plates with ampicillin. Only cells that
successfully bound the plasmid-coated nanoparticles through the
aptamer-molecular target link and transferred an expressible
plasmid to the recipient parental host will grow on the ampicillin
medium. The surviving clones can be selected and amplified by
further culturing in medium. DNA can be extracted from this culture
and the aptamer can be cut out of the plasmid and sequenced to
reveal the specific aptamer selected. When these surviving clones
are grown on 3-AT medium, they will produce an organic
semiconductor (e.g. diazoluminomelanin (DALM)) which will be
biosynthetically linked through the plasmid DNA containing the
specific aptamer to the target microbe. The complex can be
incubated with a target molecule-containing sample. Then,
activation with ultraviolet (UVA or UVB) light, sodium bicarbonate
and hydrogen peroxide and heat or microwave energy, the target
molecule will be killed or neutralized. Alternatively, these
DALM/plasmid/aptamer complexes can be used also to detect and
identify the target microbe by specific binding detected by
thermochemiluminescence, slow fluorescence, visible light
absorption, or colorimetric means. The magnetic property of the
nanoparticles, which bind non-covalently to the DALM/plasmid/DCE,
can be used to concentrate the complex bound to the target for
detection, identification, or further analysis. Finally, the
DALM/plasmid/aptamer complex can be used to target the free-radical
destruction of target agent facilitated by microwave radiation, UV
and visible light, heat, and chemical agent exposure.
Example 3
[0124] In another exemplary method, freeze-dried seed stock library
of 1MI 09/pIC20R NR1.1DCE (E. coli host containing a library of
random aptamer/DCE sequences in the plasmid with the genes for
ampicillin resistance and the capability to make organic
semiconductor) can be grown up in liquid growth medium using the
standard portable field incubator used currently in the USAF for
water quality assessment (coliforms). This culture would form the
stock of plasmid, following lysis with a lysis solution, for
selecting the new DNA aptamer sequences for a biological agent
discovered in the field. The biological agent source of interest
would then be mixed with metallic nanoparticles that chemically
(covalently or non-covalently) link to molecular targets on the
surface of microbes or directly to molecular-scale toxins. These
would be "purified", extracted from the milieu, and separated by an
attractant such as a magnet. The collected nanoparticle bound
target would then be washed several times with water or buffer and
then lysed with a lysing buffer or solution (similar to what was
used for the library bacteria). This latter process would kill and
break up the agent but leave the molecular target bound to the
nanoparticle. The nanoparticles would then be magnetically
extracted (same as above) and washed with a solution compatible
with the binding of the plasmid/DCE. This preparation would then be
added to a re-constituted culture of freeze-dried JMI 09 parent
(lacking plasmid). After the appropriate time, the bacteria would
be deposited on a culture filter (similar to those used in the
standard water quality assay) and placed over a sponge containing
the ampicillin-containing culture medium. This preparation is then
incubated in the portable incubator until colonies appear on the
filter grid. Isolated colonies can be picked off with a sterile
swab or loop and transferred to culture medium for expanding the
clones. These clones are the ones that will make DCE against the
new target on demand if they are grown in 3AT liquid medium in the
portable incubator and the DALM/plasmidlDCE is extracted into the
supernatant (just the spontaneous lysate are left when the solids
fall out of solution or are frozen out), this crude supernatant can
be used to detect, identify and sensitize the target biological to
killing as mentioned above. Also, the DNA from this supernatant
contains the sequence `fingerprint` that can be dried and sent for
further analysis such as sequencing.
Example 4
[0125] Some advantages of the embodiments herein are that selected
aptamers of the selected plasmid-random aptamers are a signature of
a target molecule of interest. The sequence may match or show
homology to one in a database of aptamer sequences or it can be
used to against known agents supporting laboratories to validate
identification without the need to transport a target the agent
from the field. Therefore, the DCE/aptamer is selected and
manufactured in the field in the forward area for use against a
particular agent. All the reagents (mostly media and lysis solution
components) can be kept in un-refrigerated freeze-dried packaged
form to be reconstituted with water. The cultureware can be plastic
and disposable and carried in a cooler-sized portable incubator.
Pre-placed freeze-dried seed stocks against a given agent can be
placed in the field for manufacture of detecting, identifying and
neutralizing DCEs, similar to what is described above for selecting
DCEs for unknown agents. The non-specific linking of the
nanoparticles to the surface of an intact agent before the lysing
of the microbe also tends to select molecular targets on the
microbe that are accessible and associated with a living or viable
agent, other than culture, no existing binding method can determine
agent viability.
Example 5
[0126] An alternative selection method would be uptake of
nanoparticles coated with a molecular target into a living microbe
or cell in order for binding to the aptamer to occur
intracellularly. After binding, the microbe or cell is lysed and
the nanoparticle with the attached aptamer is magnetically
extracted. It is then added to a new parent microbe or cell which
is selected by the antibiotic resistance marker on the plasmid when
successfully incorporated. The same approach as above can be
applied to mammalian cells. Methods and compositions of plasmids
and vectors for placing and expressing organic
semiconductor-producing gene in animal and human cells and this
plasmid could potentially harbor the DCEs. Also, it contains a
neomycin-resistance gene that is expressed and allows for the
selection of gene recombinants in these cells. An E. coli HB101
host, containing this vector plasmid pSV2neoNR1O I (American Type
Culture Collection #69617; U.S. Pat. No. 5,464,768), can be used to
carry and propagate the DCE inserts. This host and plasmid could be
used to produce the DCE clones against a specific biological agent
as described above. Then, the selected plasmids could transferred
to a parent HB101 for further propagation of the plasmid and/or
transfected by the same nanoparticle carrier technique into a host
animal or human cell for the production of DALM and DCEs that would
be specifically directed against an intracellular target, through
DCE/DALM/DNA, or the RNA/DALM counterpart's binding and microwave
radiation activation of neutralization. The DNA DCEs would be
converted into RNA, previously selected against a target, or by
transcription of the RNA (intracellular) to be selected for binding
to intracellular targets. Thus, the DCEs in the plasmids would be
cDNA of the actual RNA aptamers that would attach to the
target(data not shown). DALM/DCE plasmids can successfully
transfect parental JM109 and HB101 strains of E. coli. Second
generation transfection of parental E. coli with the initial
transfectants' DALM/DCE plasmids has been successfully completed
(data not shown).
FIGURES
[0127] FIG. 1 represents interactions between nanoparticles, DNA
and organic semiconductors.
[0128] FIG. 2 illustrates plasmid generation and transfer (200) of
some embodiments disclosed herein. In this schematic, 202
represents a plasmid with aptamer (DCE) and nitrate reductase and
ampicillin resistance genes for DALM production and clonal
selection, respectively; 203 represents bacterial chromosome; 204
represents DALM nanobe "coat" and 201 represents a host microbe for
synthesizing random aptamer or select aptamer-plasmid
complexes.
[0129] A pIC plasmid vector can be represented. The Taq1 site is
used as a reference point. (at 4018 of pBR322). The other Taq1
sites are located between Hind III and the polylinker. Other sites
are shown for insertion of a selected aptamer or random aptamer
library.
[0130] FIG. 3 illustrates an exemplary eukaryotic nanobe, RAW 264.7
cells making DALM structures is represented. In this figure, 300,
305 represents the nucleus; 312 represents DNA; 306 represents
mRNA; 307 represents NR (nitrate reductase); 310 represents DALM;
308 represent aptamer RNA; 303 represents DALM; 302 represents NP
(metallic nanoparticle); and 301 represents a bacterial host and
311 represents release and binding of the aptamer-DALM complex
308/310 to intracellular targets or those in or on other cells.
[0131] FIG. 4 illustrates an example of isolation and
electrophoretic separation of DNA fragments of Nitrate Reductase
presence in HK2 cells after exposure to synthetic DALM coated
nanobe.
[0132] FIG. 5 illustrates a schematic of aptamer selection and
nanobe replication in a remote area the star represents immobilized
or captured agent; 501 represents aptamer-plasmid complex; 503
represents a nanoparticle; 502 represents DALM; 505 represents a
bacterial host and 504 represents replication of a plasmid-selected
aptamer after isolation and introduction to a host for
replication.
Materials and Methods
[0133] Some embodiments may include using one or more of the
experimental methods outlined below.
DALM Production
Solutions:
[0134] Luria Bertani (LB) agar plates: dissolve 1 LB agar tablet
(Sigma-Aldrich, L7025) per 50 mL dH2O. Autoclave at 121.degree. C.
for 15 min. Once cooled, pour the LB/AMP agar into 100 mm.times.15
mm petri-dishes (Fisher, MS-D13-00325), let solidify and store at
4.degree. C.
[0135] LB/Ampicillin (AMP) agar plates: dissolve 1 LB agar tablet
per 50 mL dH2O. Autoclave at 121.degree. C. for 15 min. Once agar
reaches 40.degree. C., replace 30 .mu.L per 100 mL LB broth with 30
.mu.L of 100 mg/mL AMP (Sigma Aldrich, A8511) stock to make [30
m/mL]. Pour the LB/AMP agar into 100 mm.times.15 mm petri-dishes,
let solidify and store at 4.degree. C.
[0136] Tryptic Soy Broth (TSB): dissolve 15 g of Tryptic Soy Broth
(MP, 101717) into 500 mL dH2O and autoclaved at 121.degree. C. for
15 minutes (min.). Once cooled, store at 4.degree. C. 4.times.3AT
MEDIA: In 4 separate 2 L glass flasks and 1 L glass bottle make 1
liter solutions of 60 mg/L of 3-Amino-L-Tyrosine dihydrochloride
(Sigma-Aldrich, A9383), 6 g/L of potassium nitrate (Fisher,
BP368-500), 50 mg/L of Luminol (Sigma-Aldrich, A8511) and 30 g/L of
TSB. Autoclave at 121.degree. C. for 15 min. When media cools to
40.degree. C., replace 300 .mu.L of media in flasks with 300 .mu.L
of 100 mg/mL AMP stock to make a [30 m/mL]. When 1 L bottle cools,
store at 4.degree. C. The following procedure may be used: Streak
strains of OAJ 6, 7, 8 and JM109 pIC (all containing the AMP
resistant plasmid insert NR1.1) onto separate LB/AMP agar plates
using 1 .mu.L inoculating loops (Fisher, 14-906-29). Streak JM109
parental strain (no NR1.1 plasmid insert) onto an LB agar plate and
an LB/AMP plate (as a negative control validation). Place plates in
a 37.degree. C. incubator overnight. After overnight incubation,
observe plates for growth. There should be no growth of JM109
parental strain on LB/AMP plates. Then, Pipet 12 mL of TSB in a 15
mL conical Falcon tube (Fisher, 14-959-70 C). Replace 3.64, of
media with 3.64, of 100 mg/mL AMP stock. After vortexing, pipet 3
mL into 4 separate 5 mL snap cap tubes (Fisher, 14-959-2A). Pick
clones of OAJ 6, 7, 8 and JM109 pIC from LB/AMP plates and
inoculate into each properly labeled, respective 5 mL snap cap tube
and place slanted in 37.degree. C. incubator shaker overnight. Pour
liquid cultures into properly labeled and respective 2 L flasks
containing 4.times.3 AT media/AMP. Cover each flask cheesecloth
squares (Fisher, AS-240) secured with tape and loosely cover with
foil secured with tape onto flask as well. Place all 4 flasks into
37.degree. C. shaker incubator for 5 days. After about 5 days time
period, make 5 mL aliquots in 15 mL conical Falcon tubes, labeled
accordingly (OAJ 6, 7, 8 and JM109 pIC) and place in -20.degree. C.
freezer overnight. Once solidified, a top layer is observed in the
15 mL conical Falcon tubes. Open tubes and invert onto
petri-dishes. Only the top layer is allowed to melt and collect in
the petri-dishes while the remaining contents of the 15 mL tube are
properly discarded. Collect the top layer, synthesized DALM, into
separate 50 mL conical Falcon tubes (Fisher, 14-959-49 A), properly
labeled OAJ6, OAJ7, OAJ8 or JM109 pIC, cover with foil and store at
4.degree. C.
Coating Nanoparticles (Fe) with DALM Cocktail
[0137] In some embodiments, nanoparticles may be coated with an
organic semiconductor. In one example, nanoparticles are coated
with DALM. A stock of nanoparticles, 12.54 of [10 .mu.g/.mu.L]
stock of various nanopaticles (Fe, Ag, FeAgC and FeC), is mixed
with 500 .mu.L DALM cocktail in respective 2.0 mL microcentrifuge
tubes. Then, wrap each tube in foil and allow to incubate at
4.degree. C. with constant rotation overnight. After overnight
incubation, use a magnetic microcentrifuge chamber to separate the
nanoparticles from the DALM. With the nanoparticles collected on
one side of the tube, extract the 500 .mu.L DALM carefully from the
tube using a transfer pipet and replace it with 500 .mu.L of dH2O.
Reconstitute the nanoparticles in the 5004 dH2O by vortexing.
Repeat wash steps twice more for each tube to ensure the
nanoparticles are washed completely. After the final extraction of
dH2O, wrap tube in foil and store at 4.degree. C. until needed.
JM109 parental strain exposed to DALM-Coated Fe nanoparticles
(DCFe)
[0138] Solutions: Fe nanoparticles (NP)--using proper PPE, weigh
out 100 mg of 15 nm Fe NP (e.g. Nanotechnologies, Fe-15-ST2,
M19003) and suspend in 10 mL of chilled dH2O in a glass
scintillation vial (Research Products Intl, 3002-1RP). Make [10
.mu.m/.mu.L] stock solution. Directly sonicate (Branson Sonifier,
Model 250) solution for 5 minutes in 1 minute intervals to keep NP
in solution. Store at 4.degree. C.
[0139] DALM cocktail--combine 300 .mu.L of each strain of
synthesized DALM into a 5 mL snap cap tube.
[0140] LB Broth--place 1 LB broth tablet (Sigma-Aldrich, L7275),
per 50 mL of dH2O. Autoclave at 121.degree. C. for 15 min. Store at
4.degree. C.
[0141] LB/AMP Broth--Replace 30 .mu.L per 100 mL LB broth with 30
.mu.L of 100 mg/mL AMP stock to make [30 m/mL] concentration. Make
12-10 .mu.L aliquots of Fe NP stock solution in separate 0.5 mL
microcentrifuge tubes (USA Scientific, 1605-0000) and store in
4.degree. C. After vortexing the DALM cocktail, pipet 200 .mu.L
into 4 of the Fe NP aliquot stocks. Cover the tubes with foil,
place on a rotator and incubate at 4.degree. C. overnight. After Fe
NP are coated with DALM cocktail, uncover tubes and wash three
times with dH2O using a magnetic microcentrifuge tube holder
(Dynal). Once washed, pipet 200 .mu.L of LB broth into tubes and
vortex. Label 4-15 mL conical Falcon tubes JM109 1DCFe and fill
with 4.8 mL of LB broth. Pipet 200 .mu.L of LB/DCFe NP in
microcentrifuge to 15 mL tubes and vortex. Pick a clone of JM109
parental from LB agar plate and inoculate each 15 mL Falcon tube.
Loosely cover the tubes and place slanted in 37.degree. C. shaker
incubator for 12-16 hr. Streak 1 mL of each liquid culture in 200
.mu.L aliquots onto LB/AMP plates and incubate at 37.degree. C.
overnight. Pick 4 clones from LB/AMP plates and inoculate 5 mL
LB/AMP broth in separate properly labeled 15 mL conical Falcon
tubes. Loosely cover the tubes and place slanted in 37.degree. C.
shaker incubator for 12-16 hr. Also, pipet 5 mL LB/AMP broth into
properly labeled 15 mL conical Falcon tubes and inoculate separate
clones of OAJ6, OAJ7, OAJ8 and JM109 pIC. Loosely cover the tubes
and place slanted in 37.degree. C. shaker incubator for 12-16
hr.
DNA Extraction: Boiling Miniprep
[0142] Solutions: STET: for 200 mL; 16 g sucrose (Sigma, S1174), 10
mL 1M TrisCl (??), pH 8.0, 20 mL 500 mM EDTA (Invitrogen,
15575-020), 10 mL Triton X-100 (Sigma, T9284) and add dH20 to 200
mL. Filter sterilized and stored at 4.degree. C.
[0143] Lysozyme: 10 mg Lysozyme powder (Sigma-Aldrich, L-7651)
dissolved in 1 mL TE (Invitrogen, 12090-015). Make fresh each time
and kept on ice until used. 8M ammonium acetate: 61.66 g of
ammonium acetate (Sigma, A1542) dissolved qs to 100 mL dH2O. Filter
sterilized and stored at room temperature (temp). Ammonium
acetate/isopropranol: 5 volumes of 8M ammonium acetate to 12
volumes of Isopropanol (Sigma, 19030). Mix and store in sterile
bottle at room temp. Do not autoclave solution.70% ethanol: Mix 70
mL absolute ethanol and 30 mL dH2O, Store in sterile bottle at
4.degree. C.
[0144] After vortexing LB/AMP liquid cultures grown overnight,
transfer 2.0 mL into separate, properly labeled 2.0 mL
microcentrifuge tubes (Fisherbrand, 05-408-138) and centrifuge
(Eppendorf Centrifuge, 5417R) at 5000 rpm for 4 minutes. A blank
with 2 mL of dH2O will also need to be made. Aspirate supernatant
and pipet 300 .mu.L of STET to pellets in tubes and vortex to
reconstitute cells in solution. Add 25 .mu.L of Lysozyme solution
into each tube and vortex. Immediately transfer tubes to
100.degree. C. heating block for 2 minutes. Centrifuge tubes at
16400 rpm for 5 minutes at room temperature. Discard loose pellet
using a sterile inoculating loop and add 300 .mu.L of ammonium
acetate/isopropanol solution to the supernatant in each tube and
vortex. Centrifuge tubes at 14000 for 5 minutes at room
temperature. Pour off supernatant and blot tubes on paper towel.
Add 500 .mu.L of 70% ethanol to pellet, vortex and centrifuge tubes
at 16400 rpm for 5 minutes at room temperature. Pour off
supernatant and blot tubes on paper towel and allow pellet and tube
to completely dry before adding 50 .mu.L of TE buffer.
DNA Extraction: Spin Preparation
[0145] Qiaprep Spin: Overnight liquid cultures (1.5 mL of the 2 mL)
were transferred to respective 2 mL microcentrifuge tubes (USA
Scientific, 1480-2700) and centrifuged a 8000 rpm for 3 min
(Eppendorf, Centrifuge 5417R. After the tube was inverted 4 times,
3504 of Buffer N3 was mixed immediately and thoroughly by inverting
the tubes 4 times. The tubes were centrifuged for 10 min at 13,000
rpm. The supernatant of each tube was then transferred to the
QIAspin column by decanting and centrifuge for 1 min at 13,000 rpm.
After the flow-through was properly discarded, the columns were
washed with 5004, of Buffer PB and centrifuged for 1 min at 13,000
rpm. The columns were washed with 7504 of Buffer PE and centrifuged
for 1 min at 13,000 rpm. The tubes were centrifuged for 1 min at
13,000 rpm to remove any residual wash buffer. The columns were
transferred to a clean 2.0 mL microcentrifuge tube and 504 of
buffer EB was added. The columns were allowed to stand for 1 min
before being centrifuged for 1 min at 13,000 rpm. The DNA was then
properly stored in -20.degree. C. freezer until needed.
[0146] In one example, fragments of interest may be amplified and
detected. In this example, NR1.1 was amplified and isolated.
Detection via PCR and Electrophoresis
[0147] PCR Master Mix: 5 .mu.L-10.times.PCR Rxn Buffer (e.g.
Invitrogen, Y02028); 2 .mu.L-50 mM MgC12 (e.g. Invitrogen, Y02016);
1.3 .mu.L-5% DMSO (e.g. Sigma, D2650); 1 .mu.L-dNTP (e.g. Atlanta
Biologicals, 362275); 0.4 .mu.L-Taq polymerase (e.g. Invitrogen,
18038-042), added last 20.3 .mu.L-dH2O nuclease free (e.g. Gibco,
10977-015); 2.5 .mu.L-NR1.1F primer (e.g. Sigma-Genosys,
37998580-010); and 2.5 .mu.L-800R primer (e.g. Sigma-Genosys,
37998580-020). Add 334, of Master Mix to properly labeled PCR tubes
(e.g. Fisher, 08-408-229) containing 10 .mu.L of respective DNA
extraction samples. For PCR in a thermocycler (e.g. Biometra,
T3000) as follows: 40 cycles Lid Temperature-103.degree. C.
Temperature 94.degree. C.-10 min. Temperature 94.degree. C.-45 sec.
Temperature 55.degree. C.-35 sec. Temperature 72.degree. C.-2 min.
Temperature 72.degree. C.-5 min. Temperature 4.degree. C.-pause.
Make a 1.5% agarose gel as follows: dissolve 1.5 g of pure agarose
(e.g. Invitrogen, 15510-027) into 100 mL of lx TAE Buffer (e.g.
BioRad, 161-0743). Heat in microwave on high for 1 minute and
observe agarose is completely dissolved. Pour agarose/1.times.TAE
into gel canister (e.g. BioRad) in gel mold (e.g. BioRad) with
combs (e.g. BioRad) and set aside until solidified. Once PCR is
complete, pipet 5 .mu.L, of SYBR Gold (e.g. Invitrogen, S11494) and
5 .mu.L, of sample into a separate, respective PCR tube. For Ladder
(e.g. Fermentas, SM1373): pipet 5 .mu.L, SYBR Gold and 2 .mu.L of
Ladder. Carefully pipet all contents of each tube into respective
well and run electrophoresis unit (BioRad, Model 200) for 70V for
30 minutes. View gel using UV light source (Syngene, G-Box) and
observe 1.1 kb bands (e.g. NR1.1).
Synthesis of 1st Generation Transformation DALM
[0148] Streak 1 mL of each remaining JM109 DCFe (DALM coated iron
nanoparticle) liquid cultures onto properly labeled and respective
LB/AMP agar plates in 2004, aliquots and incubate at 37.degree. C.
Pipet 12 mL of TSB in a 15 mL conical Falcon tube and replace 3.6
.mu.L of media with 3.64, of 100 mg/mL AMP stock. After vortexing,
pipet 3 mL into 4-5 mL snap cap tubes. Pick 4 clones from LB/AMP
agar plates and inoculate the 4-5 mL snap cap tubes containing
TSB/AMP. Place tubes slanted in 37.degree. C. shaker incubator
overnight. In 4-2504 glass flasks, pour 100 mL of 4.times.3 AT
media and replace 304 of media with 304 of 100 mg/mL AMP stock to
make [30 .mu.g/mL]. Pour TSB/AMP liquid cultures into a separate
flask containing 4.times.3 AT/AMP and cover flasks with cheesecloth
squares secured with tape and loosely cover with foil secured with
tape. Place flasks in 37.degree. C. shaker incubator for 2-3 days.
After incubation period, make 5 mL aliquots in 15 mL conical Falcon
tubes, labeled accordingly (e.g. JM109 DCFe) and place in
-20.degree. C. freezer overnight. Once solidified, a top layer is
observed in the mL conical Falcon tubes. Open tubes and invert onto
Petri-dishes. Only the top layer is allowed to melt and collect in
the petri-dishes while the remaining contents of the 15 mL tube are
properly discarded. Collect the top layer, synthesized DALM, into a
50 mL conical Falcon tubes labeled JM109 1DCFe DALM, cover with
foil and store at 4.degree. C.
JM109 parental strain exposed to DCFe of 1st Generation
Transformation
[0149] Obtain (e.g. 4) Fe NP aliquot stocks and pipet 200 .mu.L of
JM109 1DCFe DALM into each microcentrifuge tube, cover with foil,
place on a rotator and incubate overnight at 4.degree. C. After Fe
NP are coated with DALM cocktail, uncover tubes and wash three
times with dH2O using a magnetic microcentrifuge tube holder. Once
washed, pipet 200 .mu.L of LB broth into tubes and vortex. Label
4-15 mL conical Falcon tubes JM109 2DCFe and fill with 4.8 mL of LB
broth. Pipet 200 .mu.L of LB/DCFe NP in microcentrifuge to 15 mL
tubes and vortex. Pick a clone of JM109 parental from LB agar plate
and inoculate each 15 mL Falcon tube. Loosely cover the tubes and
place slanted in 37.degree. C. shaker incubator for 12-16 hrs.
Streak 1 mL of each liquid culture in 2004 aliquots onto LB/AMP
plates and incubate at 37.degree. C. overnight. Pick clones (e.g.
4) from LB/AMP plates and inoculate 5 mL LB/AMP broth in separate
properly labeled 15 mL conical Falcon tubes. Loosely cover the
tubes and place slanted in 37.degree. C. shaker incubator for 12-16
hr. Extract DNA plasmid and detect NR1.1 of 2nd generation
transformation using the protocols: DNA Extraction: Boiling
Miniprep and NR1.1 Detection via PCR and Electrophoresis. FIG. 6
illustrates an electrophoresis gel showing NR1.1 bands of 1st and
2nd generation JM109 parentals exposed to DALM-coated nanoparticles
(Fe).
[0150] FIG. 6 illustrates an electrophoresis gel illustrating JM109
parental, JM109 pIC, 1st generation of DALM-coated Fe nanoparticle
exposure and 2nd generation DALM-coated Fe nanoparticle exposure
screened for the presence of NR1.1 (AMP resistant, nitrate
reductase gene) as shown by asterisk (*). Primers Inner 1 Forward
and Reverse and Inner 2 Forward and Reverse were also used to
determine presence of NR1.1 within the gene. To obtain 3rd
generation transformation, use clones of successful 2nd generation
transformation and follow the protocols: Synthesis of 1st
Generation Transformation DALM and JM109 parental strain exposed to
DCFe of 1st Generation Transformation using DALM synthesized from
2nd generation clones. FIG. 7 represents an electrophoresis gel
showing the presence of NR1.1 bands (*) in 3rd generation
transformation of JM109 parent cells via DALM-coated Fe
nanoparticles.
RPMI 1640 Medium
[0151] Using aseptic technique, under biological hood, 55 mL of
RPMI 1640 Medium (Invitrogen, 0030078DJ) was extracted and pipetted
into a properly labeled 50 mL conical Falcon tube and stored at
4.degree. C.
MH-S Cell Line Propagation
[0152] MH-S cells (ATCC, CRL-2019), epithelial human lung
macrophage, were grown in 150 cm.sup.3 tissue culture flasks
(Fisher, 10-126-34) cultured in sterile-filtered RPMI 1640 Medium
(Invitrogen, 003-0078DJ) supplemented with 3.5 .mu.L/L of
2-mercaptoethanol (Sigma, M7522). Cells were kept in 37.degree. C.
incubator with 5% carbon dioxide atmosphere and subcultured to a
1:6 ratio when 85-90% confluent. Medium was refreshed every 2 to 3
days.
MH-S Subculturing
[0153] Remove medium from tissue culture flask, properly discard
and replace with 4 mL of Trypsin-EDTA (Invitrogen, 25200-072) to
disperse cells. Place flask in 37.degree. C. incubator with 5% CO2
atmosphere for 5 min. Observe cells under an inverted microscope to
ensure cell layer is dispersed. Add 10 mL of RPMI 1640 medium to
inhibit Trypsin-EDTA and aspirate cells by gently pipetting.
Transfer cell suspension into a 50 mL conical tube and centrifuge
(Beckman Coulter, Allegra 6R) at 15.degree. C., 2500 rpm for 5 min.
Decant supernatant properly and resuspend cells in 12 mL of fresh
RPMI 1640, gently aspirating cells. Place 2 mL into each of the 6
properly labeled tissue culture flasks containing 4 mL of medium
and place flasks in 37.degree. C. incubator with 5% CO2
atmosphere.
MH-S Preservation/Freezing
[0154] Remove medium from tissue culture flask, properly discard
and replace with 4 mL of Trypsin-EDTA (Invitrogen, 25200-072) to
disperse cells. Place flask in 37.degree. C. incubator with 5% CO2
atmosphere for 5 min. Observe cells under an inverted microscope to
ensure cell layer is dispersed. Add 10 mL of RPMI 1640 medium to
inhibit Trypsin-EDTA and aspirate cells by gently pipetting.
Transfer cell suspension to 50 mL conical Falcon tube and
centrifuge at 15.degree. C., 2500 rpm for 5 min. Discard
supernatant and resuspend cells in fresh RPMI 1640 medium (950
.mu.L per 150 cm.sup.3 tissue flask). Pipet 50 .mu.L of DMSO
(Sigma, C6295) into each properly labeled cryongenic vial (Nalgene,
5005-0015) for each tissue culture flask and pipet 950 .mu.L cell
suspension into vial. Store vial in liquid nitrogen vapor phase
after placing cell stock in a-80.degree. C. freezer overnight.
MH-S Harvesting and Cell Concentration
[0155] Following the protocol of MH-S Subculturing, after adding
fresh medium in step 6, collect the mixture from all respective
flasks in a 50 mL conical tube and centrifuge at 15.degree. C.,
3000 rpm for 5 min. Discard supernatant and pipet 1 mL of medium to
cells to determine cell concentration. Pipet 900 .mu.L of medium
and 100 .mu.L of the 1 mL cell/medium mixture, previously
mentioned, into a 15 mL conical tube and pipet 10 .mu.L of that
into a hemocytometer (Fisher, 0267110). Each grid of the
hemocytometer represents a total volume of 0.1 mm3 or 10.sup.-4
cm.sup.3. Since 1 cm.sup.3 is equivalent to approximately 1 mL, the
total number of cells per mL was determined using the following
calculations: Cells/mL=average cell count per grid.times.dilution
factor.times.104. This value is used to determine starting
concentrations for confluency testing and cytotoxicity assays.
Confluency Testing
[0156] Using the value of cell concentration, previously mentioned
above, starting concentrations of 1.04, 3.04, 5.04, and 7.04
cells/mL were placed into six designated wells of 24 well Falcon
culture plates (Fisher 353047) data not shown. Plates were
incubated at 37.degree. C. with an air atmosphere of 5% CO2 and
evaluated for confluency at 24, 48 and 72 hours time intervals.
After incubation periods, each well was observed for confluency
percentage on an inverted microscope. The average percentage of
respective concentrations for each time point was used in
determining starting concentrations for the cytotoxicity assays
which followed.
MH-S Confluency
[0157] 72 hrs-4.5.times.10.sup.4; 48 hrs-7.5.times.10.sup.4; 24
hrs-1.5.times.10.sup.5
XTT Cell Proliferation Assay
[0158] For each 24-well tissue culture plate, combine a 56 .mu.L
aliquot of the electron coupling reagent with a 2.8 mL aliquot of
the XTT labeling reagent and vortex well. Pipet 100 .mu.L into all
wells and place back in 37.degree. C. incubator with a 5% carbon
dioxide atmosphere for 30 min. After the 30 min time interval, the
plate was read at 490 nm (Bio-TEK, Synergy HT).
MH-S Exposure to DALM Cocktail
[0159] Harvest MH-S cells using phenol-free medium onto 24-well
tissue culture plates with the respective cell concentration/per
well with the exception of an empty "blank" well data not
shown.
MH-S Exposure to Various Nanoparticles
[0160] A 1 .mu.g/.mu.L stock of the following nanoparticles was
tested: Fe, Ag, FeAgC and FeC. In respective 15 mL Falcon tubes
containing 5 mL of phenol-free RPMI 1640 medium, 10 .mu.L of each
stock is mixed. As mentioned earlier in MH-S Exposure to DALM
Cocktail, expose the confluent MH-S cells to 1 mL the respective
nanoparticle-medium mix, data not shown. MH-S Exposure to DALM
Coated Nanoparticles. Inverted microscopic images are represented
of MH-S cells "taking up" the DALM-coated Fe nanopaticles at
40.times., FIGS. 8C and 8D, and 100X, FIGS. 8A and 8B. Post
exposure, conduct a DNA extraction of cells in the tissue culture
flasks using the Qiagen Prep mentioned earlier and HeLa NR1 cells
(ATCC, CRL-13011) as a positive control. FIGS. 9A and 9B represent
an electrophoresis gel of these exemplary methods. FIG. 9A
illustrates an electrophoresis gel of HeLa NR1 (positive control)
and MH-S macrophages exposed to DALM-coated Fe nanoparticles and
FIG. 9B represents the same gel as (A) only brighter to show bands
clearer.
Pretreatment of MH-S with DALM prior to Ampicillin Exposure
[0161] FIG. 10 illustrates an exemplary graph of a comparison of
MH-S cells exposed to phenol-free medium (Control), phenol-free
medium containing 5 mg/mL concentration of ampicillin (MH-S) and
phenol-free medium containing 5 mg/mL concentration to ampicillin
after a 24 hour pretreatment with DALM-coated nanoparticles. Here,
the control is the same as MHS (phenol-free medium).
Random Aptamer Generation
[0162] In one example, a PCR chain termination step involved
addition of 6.6 .mu.g of random (N) 60mer as a self-priming (due to
partial hybridization) PCR template with 8 .mu.g of each
deoxy/dideoxynucleotide (e.g. d/ddA, d/ddC, d/ddG, d/ddT) and 20
.mu.l (80 units) of Taq polymerase per tube. The tubes were PCR
amplified using the following temperature profile: 96.degree. C.
for 5 min, followed by 40 cycles of 96.degree. C. for 1 min,
25.degree. C. for 1 min, and 72.degree. C. for 1 min. PCR extension
was completed at 72.degree. C. for 7 min and tubes were stored at 4
to 6.degree. C. until electrophoresed. The collection of aptamers
present as overlapping random (N) 60mers or as ligated and
truncated aptamers constituted a library of aptamers.
[0163] For both types of DNA arrays, 3.3 .mu.g (typically 5 to 10
.mu.l) of library DNA was diluted with 2.times.. loading buffer and
loaded into each well of precast 10% or 4-20% gradient mini TBE
polyacrylamide gels and electrophoresed in cold 1.times. TBE for 1
h at 100 V per gel. If DNA was to be visualized in the gel, gels
were stained with 0.5 .mu.g/ml ethidium bromide in TBE for 10 min,
followed by rinsing in deionized water for 30 min and photography
on a 300 nm ultraviolet transilluminator using Polaroid type 667
film.
[0164] Arrays of aptamers can be generated from library DNA
separated by electrophoresis (size and charge). Analyte binding and
nucleic acid hybridization to the aptamer arrays can be assayed as
follows: Gels were cut into strips containing the one-dimensional
DNA arrays of either type and were added to 10 ml of BB. Gel strips
were allowed to equilibrate in their respective buffers for 10 min
at room temperature (RT) with gentle shaking and were then scanned
as described below prior to addition of analytes. All DNA analytes
were added at a final concentration of 5 .mu.g/ml and all protein
analytes were added at a final concentration of 10 .mu.g/ml in BB
for 1 hr at RT with gentle shaking Gels were gently rinsed twice in
10 ml of BB, carefully repositioned and rescanned on a luminescence
spectrometer.
[0165] In one example, a Perkin-Elmer (Beaconsfield,
Buckinghamshire, UK) model LS 50B luminescence spectrometer
equipped with a plate reader was used in the thin layer
chromatography (TLC) plate mode to scan DCE arrays in gel slices
before and after addition of various analytes. After minor swelling
or shrinkage in each of the reaction buffers, gel strips were
generally 95 to 96 mm in length, with the DNA array being contained
in the lower most 65 mm of each gel strip. Gel strips were scanned
with an excitation of 260 nm (10 nm slits), emission of 420 nm (10
nm slits) and 1 mm resolution (i.e., scanned in 1 mm increments).
ready for next aptamer selection. The aptamer/bio-agent/magnetic
bead complex can be washed thoroughly with water. The aptamer of
aptamer/bio-agent/magnetic bead complex can then be amplified by
PCR or other suitable procedure. The amplified aptamers are cloned
and then the aptamer can be sequenced.
[0166] FIG. 11 represents the fluorescence of fluorophore (or
Q-Dots) quenched by SWCNT (single-walled carbon nanotube) before a
SWCNT/primer/(DNA)aptamer/fluorophore complex penetrates a cell.
After the complex penetrates a cell membrane, the DNA (aptamer) can
be denatured from a primer, and the fluorescence detectibility
returns. The microscope images illustrates the fluorescence (e.g.
Nikon ECLIPSE E800). As used herein "fluor" can be a
"fluorophore."
[0167] Dequenching of Double-Stranded DNA Capture Element (Aptamer)
Fluor on Carbon Nanotube by Addition of Target (known to bind to
aptamer FIG. 12 represents fluorescent change after bacterial
spores (e.g. Bt and Ba) are introduced into an quenching system
(e.g. Bt aptamer). In this example, a SWCNT was used as quencher.
The microscope images demonstrate fluorescence after spores (e.g.
Bt) were introduced into the quenching system. The primary strand
of Bt aptamer is connected to carbon nanotube. The fluorophore
(Hylite 488) is connected to complementary strand. The two strands
of aptamer are annealed together. Fluorescent Change of Bt DCE
after Bt and Ba spores introduced into the system (Single-wall
carbon nanotube as a quencher, fluorophore wavelength is 529 nm).
The fluorescent intensity rises dramatically upon binding of the
target agent.
[0168] Aptamer Transfection of Mouse Macrophages (MH-S) using
Carbon Nanotubes with Double-Stranded Artificial DNA Hybridized to
Primer Complementary Strand. FIG. 13 represents a gel separation of
various constructs. Band from left to right: 1. DNA ladder; 2 and
4. Bt aptamer as control; 3, 5, and 6. sample comes from: (a)
interaction of SWCNT/primer/ss-aptamer/fl with Macrophages, (b).
the SWCNT/primer/aptamer-fl-Cell of Microphages was treated with
DNase; (c). The DNased of SWCNT/primer/aptamer/fl-Cell of
Microphages was treated with RIPA buffer; 7. Macrophage cell as
negative control 4 comes from 2 (e.g. second round PCR), 5 comes
from 3 (e.g second round PCR), and 6 comes from 5 (e.g third round
PCR).
[0169] Dequenching of Fluorochrome/Carbon Nanotube Double-Stranded
DNA Aptamer Conjugates in Eukaryotic Cells. FIG. 14 represents
microscopic images illustrating fluorescence after (1). Interacting
of SWCNT/primer/aptamer/FL with Microphage cell (2). Interacting of
SWCNT/ds-aptamer/FL with BS-C-1 cell and indicate that
SWCNT/primer/aptamer/FL complex and SWCNT/ds-aptamer/FL complex
penetrate the cell membrane and the ds-DNAs were denatured.
[0170] HeLa-P6 cell line with SWCNT/dsDNA-aptamer/Q-dots FIG. 15
represents microscopic images illustrating fluorescence after
interaction of HeLa-P6 cell line with SWCNT/ds-aptamer/Q-dots
complex and indicate that the ds-aptamer was denatured after the
complex penetrates the cell membrane.
Interaction of BS-C-1 Cell Lines with
SWCNT/ds-Aptamer/Fluorophore
[0171] Carbon Nanotubes with DNA, Fluor, in Cells FIG. 16
represents (1) microscopic images (left side) illustrating
fluorescence after interaction of BS-C-1 cell line with
SWCNT/ds-aptamer/FL complex and indicate that the ds-aptamer was
denatured (right section) after the complex penetrates the cell
membrane. Undenatured (left section) SWCNT/ds-aptamer/FL complexes
did not demonstrate fluorescence which is quenched by SWCNT (2).
Microscope images (right side) illustrate fluorescence after
interaction of HeLa-P6 cell line with SWCNT/ds-aptamer/FL complex
and indicate that the ds-aptamer was denatured after the complex
penetrates the cell membrane. Interactions of BS-C-1 and hela-P6
cell lines with SWCNT/ds-aptamer/fluorophore. (1). 10 .mu.L of
SWCNT/ds-aptamer/fluorophore are added in 150 .mu.L of BS-C-1 cell
lines (in media). The mixture is shaken at 37.degree. C. for 2 days
(Incubator). 54 of the mixture is saved for PCR (see the image of
electrophoresis of the sample) and 1 .mu.L is used for microscope.
The microscope image of BS-C-1 cell/SWCNT/ds-aptamer/fluorophore
complex is at 530 nm wavelength (top). The microscope image of
BS-C-1 cell/SWCNT/ds-aptamer/fluorophore complex at range of
visible wavelength (bottom).
Most of Fluorescenceis Quenched by SWCNT
[0172] The BS-C-1 cell/SWCNT/ds-aptamer/fluorophore complex is spun
down and washed with sterile H2O three times. The washed BS-C-1
cell/SWCNT/ds-aptamer/fluorophore complex is re-suspended in 200
.mu.L of sterile H2O and 20 .mu.L of DNase (212 unit/.mu.L,
Invitrogen) are added. The mixture is shaken at R.T for 2 hours and
the DNased BS-C-1 cell/SWCNT/ds-aptamer/fluorophore complex is spun
down and washed three times with H.sub.2O. The washed DNased BS-C-1
cell/SWCNT/ds-aptamer/fluorophore complex is re-suspended in 200
.mu.l of H2O and 5 .mu.L are saved for PCR (see the image of
electrophoresis of the sample). (3). The DNased BS-C-1
cell/SWCNT/ds-aptamer/fluorophore complex (from (2)) is spun down
and 1004 of RIPA buffer are added. The mixture is shaken at
37.degree. C. over weekend. 5 .mu.l of the mixture are saved for
PCR (see the image of electrophoresis of the sample). Interaction
of Hela-P6 cell lines with SWCNT/ds-aptamer/fluorophore (1). 10
.mu.l of SWCNT/ds-aptamer/fluorophore are added in 150 .mu.l of
Hela-P6 cell lines (in media, from Melanie). The mixture is shaken
at 37.degree. C. for 2 days (Incubator). 5 .mu.L of the mixture is
saved for PCR (see the image of electrophoresis of the sample) and
1 .mu.L is used for microscope. The microscope image of HeLa-P6
cell/SWCNT/ds-aptamer/fluorophore complex is represented at range
of visible wavelength (top). The microscope image of HeLa-P6
cell/SWCNT/ds-aptamer/fluorophore complex is represented at 530 nm
wavelength (bottom). (2). The HeLa-P6
cell/SWCNT/ds-aptamer/fluorophore complex is spun down and washed
with sterile H.sub.2O three times. The washed HeLa
cell/SWCNT/ds-aptamer/fluorophore complex is re-suspended in 200
.mu.l of sterile H.sub.2O and 20 .mu.L of DNase (212 unit/.mu.l,
Invitrogen) are added. The mixture is shaken at R.T for 2 hours and
the DNased HeLa-P6 cell/SWCNT/ds-aptamer/fluorophore complex is
spun down and washed three times with H.sub.2O. The washed DNased
HeLa-P6 cell/SWCNT/ds-aptamer/fluorophore complex is re-suspended
in 200 .mu.l of H.sub.2O and 5 .mu.l are saved for PCR (see the
image of electrophoresis of the sample). (3). The DNased HeLa-P6
cell/SWCNT/ds-aptamer/fluorophore complex (from (2)) is spun down
and 1004 of RIPA buffer are added. The mixture is shaken at
37.degree. C. for a couple of days. 5 uL of the mixture are saved
for PCR (see the image of electrophoresis of the sample). An
electrophoresis image of HeLa-P6 cell/SWCNT/ds-aptamer/fluorophore
and BS-C-1 cell/SWCNT/ds-aptamer/fluorophore complexes is
demonstrated (data not shown). For BS-C-1
cell/SWCNT/ds-aptamer/fluorophore complex, the outside Bt aptamers
of the cell are destroyed by DNase. The gel illustrated a band
after lysis of BS-C-1 cell/SWCNT/ds-aptamer/fluorophore complex.
This band indicates that the SWCNT connected ds-Bt aptamer with
fluorophore penetrate the BS-C-1 cell. For HeLa-P6
cell/SWCNT/ds-aptamer/fluorophore complex, the sample of DNased of
HeLa-P6 cell/SWCNT/ds-aptamer/fluorophore [from (2)] illustrates
the band and the lysed sample shows no band. The interaction of
SWCNT/ds-aptamer/fluorophore complex with HeLa-P6 was repeated and
the electrophoresis illustrated the same result.
[0173] PCR Results on Carbon Nanotube/DNA/Fluor Nanobes in Cells
FIG. 17 represents that electrophoresis does not show band (band 2)
that indicates that the outside aptamers of
SWCNT/ds-aptamer/FL-BS-C-1 cell complex are degraded by DNase.
Electrophoresis illustrates band (band 3) that indicates that the
aptamers of inside cell are released after the cell membrane of
BS-C-1 is destroyed by buffer (e.g. RIPA). 1. BS-C-1
Cell/SWCNT-ds-aptamer-fluorophore complex 2. DNased of BS-C-1
Cell/SWCNT-ds-aptamer-fluorophore complex 3. Lysed BS-C-1
Cell/SWCNT-ds-aptamer-fluorophore complex.
Preparation of HeLa-P6 cell/SWCNT/ds-aptamer/Q-dots and FeO (nano
particles) complexes for TEM 10 .mu.l of SWCNT/ds-aptamer/Q-dots
and 10 .mu.l of SWCNT/ds-aptamer/FeO (nanoparticles, 15 nm) are
added in 1004 of HeLa-P6 cell line respectively. The mixtures are
shaken at 37.degree. C. for 2 days (Incubator) and saved for TEM.
FIG. 18: represents TEM images indicate of SWCNT/aptamer/DNA/iron
nanoparticle complexes that can penetrate the cell membrane.
Preparation of BS-C-1 cell/SWCNT/ds-aptamer/FeO (nano particles)
complexes for TEM
[0174] 10 .mu.l of SWCNT/ds-aptamer/FeO (nanoparticles, 15 nm) are
added in 1004 of BS-C-1 cell. The mixtures are shaken at 37.degree.
C. for 2 days (Incubator) and saved for TEM. FIG. 19 represents TEM
images indicate of SWCNT/aptamer/DNA/iron nanoparticle complexes
that can penetrate the cell membrane with arrows indicating
intracellular aggregate nanobe structures. Materials, protocols and
methods
[0175] Conjugation of single wall carbon nano tube (SWCBT) with
phosphatidyl serine; with DNA primer; with DNA primer and
complementary fluorophore labeled aptamer. The SWCNT with a
carboxyl functional group was purchased (e.g. Sun Innovation Inc.)
and phosphatidyl serine was obtained (e.g. Signa-Aldrich;
1,2-Diacyl-sn-glycero-3-phospho-L-serine; Product Name: P7769)
Preparation of SWCNT-phosphatidyl serine
[0176] (1). Covalent bonding between SWCNT and phosphatidyl serine:
about 1 mg phosphatidyl serine is added to small amount of SWCNT in
0.01M MOPs buffer and 15 mg EDC and 5 mg imidazole are added. The
reaction mixture was stirred overnight. The product,
SWCNT-phosphatidyl serine, is separated and washed with water and
re-suspended in 1 ml PBS buffer.
[0177] Non-covalent bonding between SWCNT and phosphatidyl serine:
about 1 mg phosphatidyl serine is added to small amount of SWCNT in
0.01M MOPs buffer. The reaction mixture was stirred over weekend.
The SWCNT-phosphatidyl serine (non-covalent bonding) is separated
and washed, then re-suspended in 1 ml PBS buffer.
Preparations of SWCNT-Primer, SWCNT-Primer/Aptamer and
SWCNT-Primer/Aptamer-Q-Dots
[0178] (1). About 0.5 .mu.L of primer (Forword or Reverse) with
--NH2 functional group (F: 6.25.times.10 11 DNA/.mu.L or R:
6.88.times.11 11 DNA/4) are added to a small amount of SWCNT in
0.1M MOPS buffer respectively. Then 15 mg EDC and 5 mg imidazole
are added. The mixture is stirred overnight. The SWCNT-primer (F or
R) are separated and washed, then re-suspended in TE buffer. (2).
0.5 4 of Bt aptamer (primary or complementary strand) are added to
SWCNT-Primer (F or R) respectively, (of note, the 3' side primer of
aptamer should be complementary to the primer bound to the SWCNT
and 5' side of the aptamer is --PO4 functional group). The mixture
is stirred overnight, then separated and washed. The
SWCNT-primer/aptamer is re-suspended in TE buffer.
Preparations of SWCNT/ss-Aptamer, SWCNT/ds-Aptamer and
SWCNT/ds-Aptamer/Q-Dots
[0179] (1). 10 .mu.l of Bt aptamer (complementary, --NH2 functional
group, 1.4.times.10 13 DNA/4) are added to a small amount of SWCNT
in 0.1M MOPs buffer. Then 60 mg EDC and 20 mg imidazole are added.
The mixture is stirred overnight. The SWCNT-ss-aptamer is separated
and washed, then re-suspended in 0.1M MOPs buffer. (2). 15 4 of Bt
aptamer (primary, --PO.sub.4 functional group, 1.3.times.10 13
DNA/4) are added to the SWCNT/ss-aptamer in 0.1M MOPs buffer. The
mixture is stirred at 60.degree. C. for 6 hr and R.T. overnight.
The SWCNT/ds-aptamer is separated and washed, then re-suspended in
0.1M MOPS buffer. (3). 200 .mu.l of Q-dots are added to the
SWCNT/ds-aptamer in 0.1M MOPS buffer. Then 60 mg EDC and 20 mg
imidazole are added. The mixture is stirred overnight. The product,
SWCNT/ds-aptamer/Q-dots, is separated and washed, and re-suspended
in 2 ml PBS buffer.
[0180] After PCR of SWCNT-Primer/aptamer, the image of the gel
illustrates a clear band with pure Bt aptamer as positive control.
(3). 100 ul of Q-dots (--NH2 functional group) are added to the
SWCNT-Primer/Aptamer in 0.1M MOPs buffer. Then 30 mg EDC and 10 mg
imidazole are added. The mixture is stirred overnight. The
SWCNT-Primer/Aptamer-Q-dots is separated and washed with water,
then re-suspended in PBS buffer.
[0181] Preparations of SWCNT (nanotube, quenching
agent)-ss-aptamer, SWCNT-ds-aptamer, and
SWCNT-ds-aptamer-Fluorophore 10 .mu.l of Bt aptamer (complementary,
5' side was --NH2 functional group, 1.4.times.10 13 DNA/.mu.L) are
added to a predetermined amount of SWCNT in 0.1M MOPs buffer. Then
60 mg EDC and 20 mg imidazole are added. The mixture was stirred
overnight. The SWCNT-ss-aptamer was separated and washed, then
re-suspended in 0.1M MOPs buffer. 15 .mu.A of Bt aptamer (primary,
5' side was --PO.sub.4 functional group, 1.3.times.10 13 DNA/.mu.L)
are added to the SWCNT-ss-aptamer in 0.1M MOPs buffer. The mixture
was stirred at 60 degree C. for 6 hr and R.T. overnight. The
SWCNT-ds-aptamer is separated and washed, then re-suspended in 0.1M
MOPs buffer. 15 .mu.l of fluorophore (Hylyte Fluor. 488, 1.times.10
14 molecule/.mu.L; Anaspec, Inc) are added to the SWCNT-ds-aptamer
in 0.1M MOPs buffer. Then 60 mg EDC and 20 mg imidazole are added.
The mixture was stirred overnight. The product,
SWCNT-ds-aptamer-fluorophore, was separated and washed, and
re-suspended in PBS buffer. (The fluorescence was quenched by
SWCNT. The fluorescence comes back after interaction of
SWCNT-ds-aptamer-fluorophore with Bt spores).
[0182] All of the COMPOSITIONS and/or METHODS and/or APPARATUS
disclosed and claimed herein can be made and executed without undue
experimentation in light of the present disclosure. While the
compositions and methods have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variation may be applied to the COMPOSITIONS and/or METHODS and/or
APPARATUS and in the steps or in the sequence of steps of the
method described herein without departing from the concept, spirit
and scope of herein. More specifically, it will be apparent that
certain agents which are both chemically and physiologically
related may be substituted for the agents described herein while
the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art
are deemed to be within the spirit, scope and concept as defined by
the appended claims.
* * * * *