U.S. patent application number 14/376003 was filed with the patent office on 2015-01-15 for direct detection of disease biomarkers in clinical specimens using cationic nanoparticle-based assays & versatile and green methods for synthesis of anisotropic silver nanostructures.
This patent application is currently assigned to AMERICAN UNIVERSITY OF CAIRO (AUC). The applicant listed for this patent is AMERICAN UNIVERSITY OF CAIRO (AUC). Invention is credited to Hassan Mohamed El-Said Azzazy, Kamel Abdelmenem Mohamed Eid, Bassem Samy Shenouda Guirgis, Sherif Mohamed Shawky Abduo.
Application Number | 20150017258 14/376003 |
Document ID | / |
Family ID | 48906034 |
Filed Date | 2015-01-15 |
United States Patent
Application |
20150017258 |
Kind Code |
A1 |
Azzazy; Hassan Mohamed El-Said ;
et al. |
January 15, 2015 |
DIRECT DETECTION OF DISEASE BIOMARKERS IN CLINICAL SPECIMENS USING
CATIONIC NANOPARTICLE-BASED ASSAYS & VERSATILE AND GREEN
METHODS FOR SYNTHESIS OF ANISOTROPIC SILVER NANOSTRUCTURES
Abstract
A gold nanoparticle-based assay for the detection of a target
molecule, such as Hepatitis C Virus (HCV) RNA in serum samples,
that uses positively charged gold nanoparticles (AuNPs) in solution
based format. The assay has been tested on 74 serum clinical
samples suspected of containing HCV RNA, with 48 and 38 positive
and negative samples respectively. The developed assay has a
specificity and sensitivity of 96.5% and 92.6% respectively. The
results obtained were confirmed by Real-Time PCR, and a concordance
of 100% for the negative samples and 89% for the positive samples
has been obtained between the Real-Time PCR and the developed AuNPs
based assay. Also, a purification method for the HCV RNA has been
developed using HCV RNA specific probe conjugated to homemade
silica nanoparticles. These silica nanoparticles have been
synthesized by modified Stober method. This purification method
enhanced the specificity of the developed AuNPs assay. The method
can detect a target molecule, such as HCV RNA in serum, by
employing modified silica nanoparticles to capture the target from
a biological sample followed by detection of the captured target
molecule using positively charged AuNPs. The assay is simple,
cheap, sensitive and specific. Another aspect of the invention is
anisotropic silver nanoparticles and methods of their use.
Inventors: |
Azzazy; Hassan Mohamed El-Said;
(Alexandria, EG) ; Shawky Abduo; Sherif Mohamed;
(Cairo, EG) ; Eid; Kamel Abdelmenem Mohamed;
(Sharkia Governorate, EG) ; Guirgis; Bassem Samy
Shenouda; (Cairo, EG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMERICAN UNIVERSITY OF CAIRO (AUC) |
New Cairo |
|
EG |
|
|
Assignee: |
AMERICAN UNIVERSITY OF CAIRO
(AUC)
New Cairo
EG
|
Family ID: |
48906034 |
Appl. No.: |
14/376003 |
Filed: |
January 31, 2013 |
PCT Filed: |
January 31, 2013 |
PCT NO: |
PCT/US13/24136 |
371 Date: |
July 31, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61594817 |
Feb 3, 2012 |
|
|
|
61593019 |
Jan 31, 2012 |
|
|
|
Current U.S.
Class: |
424/618 ;
420/501; 435/5; 436/94; 75/343 |
Current CPC
Class: |
C12Q 1/6816 20130101;
Y10T 436/143333 20150115; A01N 59/16 20130101; C12Q 1/6816
20130101; C12Q 1/70 20130101; B22F 1/0003 20130101; G01N 33/552
20130101; G01N 33/5308 20130101; C12Q 2523/31 20130101; C12Q
2563/155 20130101; G01N 33/553 20130101; G01N 33/587 20130101; C12Q
2565/628 20130101; B22F 9/20 20130101 |
Class at
Publication: |
424/618 ; 436/94;
435/5; 75/343; 420/501 |
International
Class: |
A01N 59/16 20060101
A01N059/16; B22F 1/00 20060101 B22F001/00; B22F 9/20 20060101
B22F009/20; G01N 33/53 20060101 G01N033/53; C12Q 1/70 20060101
C12Q001/70 |
Claims
1. A method for detecting a nucleic acid or a protein comprising:
contacting a sample suspected of containing a target molecule with
positively charged gold and/or silver nanoparticles, determining
the aggregation of nanoparticles after contacting them with the
sample, and detecting the target molecule in the sample when the
nanoparticles aggregate in comparison with a control sample that
does not contain the target molecule to be detected; wherein
aggregated nanoparticles are detected by a blue color and
non-aggregated gold nanoparticles by a red color.
2. The method of claim 1 that comprises detecting a target molecule
that is DNA.
3. The method of claim 1 comprising detecting a target molecule
that is RNA.
4.-22. (canceled)
23. The method of claim 1, wherein the nanoparticles comprise
positively charged gold nanoparticles.
24. The method of claim 1, wherein the nanoparticles comprise
positively charged silver nanoparticles.
25. The method of claim 1, wherein the nanoparticles are spherical
and have an average diameter of 12 to 40 nm.
26. The method of claim 1, wherein the nanoparticles are spherical
or spheroidal and have an average diameter of 15-18 nm.
27. The method of claim 1, wherein the nanoparticles are not
spherical.
28. The method of claim 1, wherein the nanoparticles are rod-shaped
(nanorods) having any aspect ratio.
29. The method of claim 1, wherein the nanoparticles are
star-shaped.
30.-33. (canceled)
34. A method for capturing at least one biological material of
interest comprising contacting said material with silica
nanoparticles for a time and under conditions sufficient for the
material to bind to the silica nanoparticles and optionally eluting
or recovering the material from the silica nanoparticles.
35. The method of claim 34, wherein the biological material is a
nucleic acid.
36. The method of claim 34, wherein the biological material is a
protein or a protein complex.
37.-58. (canceled)
59. The method of claim 34, wherein said captured biological
material is HCV RNA, said method comprising: processing a sample
suspected of containing HCV RNA to obtain a sample containing HCV
RNA, contacting the isolated RNA with silica nanoparticles
conjugated to a nucleic acid complementary to HCV RNA, removing
material that is not bound to the silica nanoparticles, eluting RNA
bound to the silica nanoparticles, contacting the eluted RNA with
cationic gold and/or silver nanoparticles, and detecting HCV RNA
when the cationic gold aggregate, wherein aggregation may be
determined colorimetrically by a color change from red
(substantially not aggregated nanoparticles) to blue (aggregated
nanoparticles) for the gold nanoparticles, or wherein aggregation
of cationic silver nanoparticles may be determined calorimetrically
by color change from yellow (substantially non aggregated
nanoparticles) to colorless or to white precipitate (aggregated
nanoparticles).
60. (canceled)
61. A method for synthesizing silver nanoparticles comprising:
chemically reducing a precursor silver compound in the presence of
a reducing agent and a capping agent for a time and under
conditions sufficient to produce silver nanoparticles.
62. The method of claim 61, wherein the precursor silver compound
is a silver nitrate, silver acetate, or silver chloride, the
reducing agent is ascorbic acid or dextrose, and the capping agent
is citric acid, sodium citrate, or potassium citrate.
63.-91. (canceled)
92. A nanostructure or microstructure produced by the method of
claim 61.
93.-138. (canceled)
139. A composition comprising a nanostructure or microstructure
according to claim 92 and optionally one or more pharmaceutically
or diagnostically suitable ingredients, which may be covalently or
non-covalently associated with the nanostructure, or
microstructure.
140.-141. (canceled)
142. A method for antimicrobial therapy comprising contacting a
microbe or a host cell with the composition of claim 139.
143. A method of diagnosis comprising processing or contacting a
biological sample with the composition of claim 139.
144.-165. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application 61/593,019, filed Jan.
31, 2012, and to U.S. Provisional Application 61/594,817, filed
Feb. 3, 2012, each of which is hereby incorporated by reference in
its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] Methods for detecting pathogens using cationic gold- or
silver-nanoparticles. The assays can detect amplified and/or
unamplified RNA and/or DNA from viruses, bacteria and other
organisms, such as Hepatitis C virus and mycobacterium
tuberculosis. The invention is also directed to anisotropic silver
nanoparticles and methods for synthesizing and using them.
[0004] 2. Description of the Related Art
[0005] Cationic gold nanoparticle based assays. Many molecular
diagnostic assays are commercially available for the detection of
several viral/bacterial nucleic acids (DNA/RNA) in patients' blood.
Although these assays have high sensitivity and specificity, most
of them are time consuming, labor intensive, expensive, and require
specialized equipment. Thus for the better control over infectious
diseases, especially in developing countries with limited resources
including Egypt, the development of novel diagnostic assays that
are simple, rapid, sensitive, specific and importantly
cost-effective is highly needed. Nanoparticle-based diagnostic
assays promise to meet these rigorous demands. Gold and silver
nanoparticles (AuNPs, AgNPs) are some of the most promising
nanoparticle candidates for diagnosis. They exhibit intense
absorbance and scattering properties due to surface Plasmon
resonance (SPR). The absorption cross-section of AuNPs was found to
be 10.sup.4 to 10.sup.5 folds higher than that of the strongest
absorbing Rhodamine-6G dye molecule[1]. When AuNPs come close
together, plasmon-plasmon coupling occurs leading to energy loss
and a shift in the absorbance peak maximum to a longer wavelength
and thus a change in color from red to blue occurs [1]. Moreover,
AuNP optical properties can be tuned by varying their size and/or
shape and AuNPs can be easily synthesized in different size ranges
and conjugated to biomolecules such as proteins and
oligonucleotides[1].
[0006] Developed AuNP-based molecular diagnostic assays can be
classified according to the detection signal into colorimetric,
scanometric, light-scattering, electrochemical and electrical,
quartz-crystal microbalance, Foster Resonance Energy Transfer
(FRET) and Nanometal Surface Energy Transfer (NSET), surface
enhanced Raman scattering and Laser diffraction assays[1].
Colorimetric detection methods can be further divided into
cross-linking (utilizing two oligonucleotide-functionalized AuNP
probes), non-cross linking (one probe) and non-functionalized (no
probes) using negatively charged AuNPs [1].
[0007] Positively charged (cationic) AuNPs have been used in
diagnostic assays [2-4]. Sun et al. [5] developed a microarray
assay (solid support) for gene expression analysis using cationic
AuNPs. First, target molecules are allowed to hybridize with probes
immobilized on the solid support and after washing, cationic AuNPs
are added. If the target is present, the AuNPs bind to the
negatively charged phosphate groups on the DNA target via
electrostatic attraction leading to the precipitation of nanogold
particles that can easily be detected using a flatbed scanner.
Assay sensitivity was estimated to be <2 pg of DNA [5]. Cationic
gold nanoparticles also have been used for the determination of DNA
chip quality prior to use, Hsiao et al. [6]
[0008] A significant drawback to the prior art methods which use
gold nanoparticles is that the presence of a capture probe in the
absence of a target molecule leads to aggregation of the gold
nanoparticles because the capture probes bind to the cationic gold
nanoparticles via their phosphate backbones. This increases the
percentage of false positive results because the cationic gold
nanoparticles aggregated by the capture probe in the absence of the
desired target molecule cause a color change from red to blue which
is falsely interpreted as detection of the target molecule. Prior
art methods require extra steps and labor are required to avoid
this problem. Another problem with prior methods is the requirement
for equipment that is often not available under field conditions,
such as the need to use a scanner to detect hybridization of a
target molecule to a capture probe or ligand or the requirement for
use of a laboratory spectrophotometer to quantify color change
produced by aggregation of gold nanoparticles.
[0009] Anisotropic Silver Nanoparticles.
[0010] Noble metals such as gold, platinum and silver particles
have unique physical, chemical, optoelectronic properties; have
been used in catalysis, plasmonics, photography, sensing, drug
delivery, imaging, antimicrobial, and information storage and
surface enhanced Raman scattering. They also serve as model system
for several applications such as electronic or magnetic. Eid &
Azzazy, International Journal of Nanomedicine 2012:7 1543-1550
(Mar. 19, 2012) describe hollow flower like silver nanostructures
produced by the methods disclosed herein and is incorporated by
reference.
[0011] Noble metal particles with interior hollow structures play a
significant role in drug encapsulation, controlled release of
drugs, cosmetics, nucleic acids, removal of pollutants, storage,
protection of other chemical reagents and biologically active
species. Likewise, the hollow metal particles have been used to
modulate refractive index, increase active area for catalysis,
improve a particle's ability to withstand cyclic changes in volume,
and to expand the array of imaging markers suitable for early
detection of cancer.
[0012] Properties of nanostructures depend on their size and shape
which subsequently determine their possible utilizations for
instance SERS, antimicrobial, catalytic effect have been depended
on the morphology of silver particles.
[0013] Current chemical, physical, biological methods for synthesis
of anisotropic nanostructures include the silver minor reaction,
polyol process, seed-mediated, light-mediated, and
template-directed growth, and lithographic fabrication.
[0014] Hollow nanoparticle structures can be fabricated by several
methods such as conventional hard templates, sacrificial templates,
soft templates, template-free methods. Similarly, hollow metallic
nanostructures have been fabricated by electrodeposition of thin
layer of another metal or salts followed by calcinations or
etching.
[0015] The difficulties encountered in fabricating anisotropic,
controlled hollow nanostructures include high cost, the use of
toxic chemicals, and expensive and complex laboratory equipment
requiring highly skilled technicians. Thus, there is a need for
methods of fabricating these structures that are faster, less
complicated and less expensive. Moreover, there is a need to
develop new kinds of nanostructures with different geometries that
provide useful functionalities, for example, for diagnostic or
therapeutic use or for studying plasmonic and other functional
properties of metallic nanoparticles.
[0016] One of the unique, promising tools in various application
the hollow metallic structures due to their lower density, higher
surface, enhancing their antimicrobial effects and other related
properties. Physical characteristics of hollow metallic
nanostructures that contribute to their useful diagnostic and
therapeutic properties include their low density compared to solid
nanoparticles, a relatively high surface area to mass, and their
capacity to be loaded or coated with other agents used for
diagnosis or therapy.
[0017] Similarly, the development of nanoparticles with unique
geometric shapes permits control of their physical properties,
including size, density, surface area to mass, and loading
capacity. Control of size and geometry is relevant to the
bioavailability of the nanoparticle per se or to covalent- or
non-covalent complex of the nanoparticle with other active
components, such as diagnostic or therapeutic agents. For example,
size and geometry affect the biological absorption, uptake,
targeting, and/or persistence of such nanoparticles.
[0018] As apparent from the background references, there is a need
for new methods that are environmentally sound (green), fast and
inexpensive. Such methods would not require the use of undesirable
polymers, surfactants or non-biodegradable reagents or require the
use of complex laboratory equipment, but would permit synthesis of
nanoparticles in a very short time while permitting control over
their size and geometry and parameters like density and surface
area. Ideally, such a method would provide a variety of different
kinds, sizes and geometries of nano structures.
BRIEF SUMMARY OF THE INVENTION
[0019] Gold nanoparticle based assays. The present invention solves
problems associated with the use of gold nanoparticles for
detection of target molecules. It detects unknown target molecules,
such as RNA or DNA in a biological sample in a homogenous based
solution, thus avoiding the requirement of having to hybridize a
nucleic acid in a sample (or attach other target molecules like
polypeptides) to a probe (or ligand) on a solid support. This
avoids the problem of false positives caused by aggregation of a
capture probe with the cationic gold nanoparticles because the gold
nanoparticles are not brought into contact with capture probes.
[0020] One way the invention accomplishes this is through the use
of silica or magnetic nanoparticles which are attached to a
specific probe (e.g., a capture probe for HCV RNA) or specific
capture ligand (e.g., for a specific polypeptide or other
molecule). This mode of capture differs significantly from prior
art methods that use gold nanoparticles to confirm the capture of a
target molecule. The invention uses the silica or magnetic
nanoparticles that are specifically attached to a specific capture
probe (or ligand) by a crosslinker to isolate or purify the target
molecule from a solution containing many molecules, such as a
complex mixture containing HCV RNA and many other nucleic acids.
The invention thus avoids the problem of false positives caused by
aggregation of gold nanoparticles when the target molecule is
absent from a sample. For example, cationic gold nanoparticles can
aggregate non-specifically in the presence of contaminating
non-target nucleic acids due to interaction of the phosphate
backbone of contaminating nucleic acids (i.e., non-target nucleic
acids) or capture probes, thus producing a false positive reading.
The invention avoids such false positive readings by separating the
step of capturing a target nucleic acid from its detection by
cationic gold nanoparticles. For example, the cationic gold
nanoparticles do not come into contact with a capture probe for HCV
RNA or the contaminating nucleic acids in a sample from a subject
suspected of being infected by HCV. This eliminates or
substantially reduces false positives due to aggregation of
cationic gold nanoparticles with a capture probe or a contaminating
nucleic acid in a biological sample.
[0021] Advantageously the invention provides a homogenous based
assay, which specifically detects target molecules, such as HCV
RNA, that is simple and can be used outside of a hospital or
medical laboratory (e.g., in the field) to detect the target
molecule visually by the naked eye without equipment like a
scanner. The invention also provides a method of determining the
amount of the target molecule in a sample because the color change
produced by the aggregation of cationic gold nanoparticles in the
presence of a target molecule is directly proportional to the
target concentration in a biological sample. Quantification does
not require the use of equipment often unavailable in the field,
such as the use of a spectrophotometer. It may be performed simply
by comparing the degree of color change produced by aggregation of
the cationic gold nanoparticles based on comparison with a standard
of known concentrations of target molecules. Alternatively, the
method according to the invention may be performed by measuring
color intensity using a portable spectrophotometer. In one aspect,
the invention is directed to a method for detecting a material or
molecule of interest, such as a nucleic acid, peptide, polypeptide
or a protein, comprising: contacting a sample suspected of
containing the material or molecule of interest, such as a nucleic
acid or a protein to be detected, with positively charged gold
and/or silver nanoparticles; determining the aggregation of
nanoparticles after contacting them with the sample; and detecting
the nucleic acid or the protein in the sample when the
nanoparticles aggregate in comparison with a control sample that
does not contain the nucleic acid or the protein to be
detected.
[0022] This method may be used to detect nucleic acids, modified
nucleic acids, or aptamers of nucleic acids, including DNA and RNA
molecules. Examples of nucleic acids to be detected by this method
include those selected from the group consisting of single stranded
RNA, single stranded DNA, double stranded DNA, stem loop RNA,
positive strand RNA, negative strand RNA, and double stranded RNA.
These nucleic acids may be viral nucleic acids, such as RNA from
HCV (hepatitis C virus) or nucleic acids amplified from viral
sources. The invention also contemplates detection of nucleic acids
from or amplified from non-viral sources, including detection of
nucleic acids or other kinds of molecules from bacteria,
mycobacteria, yeast, fungi, parasites, or other kinds of pathogens,
as well as nucleic acids from other sources, such as from
eukaryotic organisms including non-human animals or from
humans.
[0023] This method is not limited to detecting only nucleic acids,
but can be used to detect other kinds of molecules, such as for
detecting a peptide, polypeptide or protein. For example, it can be
used to detect a protein that is an enzyme, antigen, antibody, a
folded protein, an unfolded protein, a reduced protein, a
non-reduced protein, or a tumor marker.
[0024] The method may be applied using samples obtained from
various sources, such as from blood, plasma, serum, serum,
optionally containing EDTA, spinal fluid, saliva, urine, mucosal
secretions and other biological fluids or tissues. Samples may be
obtained from normal subjects, for example, for routine screening
or testing, or from subjects at risk for, suspected of having, or
who have been diagnosed with a particular condition, disorder or
disease. Examples of such subjects include patients having a viral
disease, undergoing treatment for a viral disease, or suspected of
having a viral disease; patients having a bacterial disease,
undergoing treatment for a bacterial or mycobacterial disease, or
suspected of having a bacterial disease and patients having a
fungal or parasitic disease, undergoing treatment for a fungal or
parasitic disease, or suspected of having a fungal or parasitic
disease. Samples may be obtained from subjects at different times
or stages of a condition, disorder or disease, such as during an
acute, chronic or occult phase. Samples may be obtained
longitudinally from a patient during the course of a disease or
during or after treatment, for example, at least 1, 2, 4, 8, 12,
16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 weeks during or after
cessation of therapy for a microbial disease, disorder or
condition.
[0025] This detection method may be practiced using positively
charged gold nanoparticles or positively charged silver
nanoparticles. These nanoparticles preferably are spherical or
spheroidal in geometry and have average diameters of 12 to 40 nm,
for example 15-18 nm or any intermediate sub-range or value within
these ranges, including 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30, 35 and 40 nm. Nanoparticles that are not spherical
may also be employed, such as rod-shaped particles (nanorods)
having any aspect ratio, star-shaped nanoparticles, or particles
having other unique geometries.
[0026] In a detection method, the aggregation of the gold
nanoparticles, which indicates detection of a positive sample, can
be detected by a blue color and non-aggregated gold nanoparticles
by a red color. A positive sample can be detected using silver
nanoparticles based on color change from yellow to colorless or to
a white precipitate.
[0027] Prior to detection, a sample may be processed, for example,
extracted, isolated, purified or concentrated prior to contacting
it with the gold and/or silver nanoparticles. One predetection
processing method contacts a sample with silica nanoparticles
conjugated to a ligand that binds to a molecular target, such as a
specific polynucleotide or polypeptide, to be detected (or that
binds to a substance, composition, solution, or aggregate
containing the target molecule. The target molecule binds to or is
adsorbed to the silica nanoparticles, can be washed or separated
from other components in a sample, and then eluted, separated or
otherwise recovered from the silica nanoparticles in a less complex
mixture or in a more concentrated or purified form.
[0028] In another embodiment, the invention is directed to a method
for capturing at least one biological material of interest
comprising contacting the material to be captured with silica
nanoparticles or another kind of suitable nanoparticle for a time
and under conditions sufficient for the material to bind to the
silica nanoparticles and then optionally eluting the captured
material from the silica nanoparticles and/or further purification
or characterization of the captured and released material.
[0029] This method may be used to capture a biological material
that is a nucleic acid, a synthetic or modified nucleic acid, a
peptide, polypeptide, protein or protein complex (e.g., protein
dimers or trimers or aggregates in which a protein is associated
with other molecules) or other biological components of interest.
The material of interest may be obtained from a virus, bacterium,
mycobacterium, yeast, fungus, parasite or other microorganism as
well as from other kinds of cells including mammalian or human
cells or samples, or from extracts or components from these
sources.
[0030] This capture method may also employ silica nanoparticles
conjugated to a ligand for the target molecule, such as a
polynucleotide sequence that is complementary to a target
polynucleotide or that is an aptamer that binds to the nucleic acid
to be detected. For example, the silica nanoparticles can be
conjugated to a ligand that binds to material containing or
associated with the nucleic acid to be detected and the method can
further involve isolating or purifying the nucleic acid from the
material bound to the silica nanoparticles. Similarly, other
materials, including polypeptides, glycopolypeptides, lipids and
carbohydrates can be captured using molecules that bind to them
which have been bound to or associated with silica
nanoparticles.
[0031] Those of skill in the art may select various ligands for use
in this capture method. Examples of such ligands include antibodies
or fragments of antibodies containing binding sites such as those
of IgA, IgD, IgE, IgG, IgM and the various subtypes of these kinds
of antibodies.
[0032] This capture method may be employ a ligand that is an
aptamer that binds to a protein, carbohydrate, lipid or other
material associated with the nucleic acid to be captured or
detected. It also may employ ligands, such as DNA or RNA or a
complex or aggregate of a nucleic acid with a peptide, polypeptide
or protein, that bind directly to a target molecule or target
material to be captured or detected.
[0033] The ligand in this capture method may comprise various
lectins, such as one or more mannose binding lectin(s), one or more
N-acetyl glucosamine(s) with or without sialic acid binding
lectin(s); one or more galactose/N-acetylgalactosamine binding
lectin(s); one or more N-acetylneuraminic acid binding lectin(s);
or one or more fucose binding lectin(s).
[0034] The ligand used in this method may be a specific probe for
the nucleic acid to be detected that is conjugated to at least one
member selected from the group consisting of iron oxide, gold,
silver, quantum dots and silica nanoparticles of different
sizes.
[0035] The method may further involve extracting the nucleic acid
from the material containing it or from the material from which it
is associated which has been bound to said ligand. It also may
comprise contacting the sample with nanoparticles selected from the
group consisting of iron oxide, gold, silver, quantum dots and
silica nanoparticles, which have been conjugated to a probe
comprising an oligotargeter sequence. For example, the sample may
be contacted with at least one oligotargeter selected from the
group consisting of SEQ ID NO: 1, 2, 3, 4, or 5.
[0036] Another specific aspect of the invention is a method for
detecting HCV RNA in a sample comprising one or more of the
following steps: processing a sample suspected of containing HCV
RNA to obtain a sample containing HCV RNA, contacting the isolated
RNA with silica nanoparticles conjugated to a nucleic acid or
aptamer complementary to HCV RNA, removing material that is not
bound to the silica nanoparticles, eluting RNA bound to the silica
nanoparticles, contacting the eluted RNA with cationic gold and/or
silver nanoparticles, and detecting HCV RNA when the cationic gold
aggregate, wherein aggregation may be determined calorimetrically
by a color change from red (substantially not aggregated
nanoparticles) to blue (aggregated nanoparticles) for the gold
nanoparticles, or wherein aggregation of cationic silver
nanoparticles may be determined calorimetrically by color change
from yellow (substantially non aggregated nanoparticles) to
colorless or to white precipitate (aggregated nanoparticles).
[0037] A specific embodiment of the invention is a method for the
capture of one or more target molecules (e.g., a specific nucleic
acid or protein biomarker) using modified silica nanoparticles.
Once captured and recovered, the biomarkers or target molecules are
detecting using unmodified cationic gold nanoparticles or cationic
silver nanoparticles. Once a biomarker is captured and contacted
with the cationic gold or silver nanoparticles, it can be detected
colorimetrically (e.g., visually) or spectrophotometrically.
[0038] The methods described herein can be applied to real clinical
specimens in either a solution assay format or in a solid support
assay format. The solution assay format starts by adding the target
to a mixture of cationic nanoparticles and phosphate buffer, in
case of positive sample the color changes within 2 minutes into
blue color while remains red in the negative samples. On the other
hand, the solid format assay starts by first immobilizing the
captured probes on the solid support, then the target is added onto
the plate and allowed to hybridize with the immobilized probes and
then washing with phosphate buffer to remove excess reagents and
targets, finally, the cationic nanoparticles are added, in case of
positive samples the color changes from red to blue while, remains
red in case of negative sample.
[0039] Another embodiment of the invention is kit comprising
positively charged gold and/or silver nanoparticles; a ligand, such
as an antibody, aptamer, lectin, or probe, which is conjugated to
iron oxide, gold, silver, quantum dots or silica nanoparticles (or
other suitable nanoparticles), and optionally, at least one
biological sample preservative, buffer or additive, a nucleic acid
extractant buffer, a reaction buffer, a negative control sample, a
positive control sample, a reaction container, a colorimetric
chart, a packaging material, guide, and/or an instruction for use
in detecting or capturing a nucleic acid or other biological
molecule.
[0040] Anisotropic silver nanoparticles: The inventors have
discovered and developed methods having these unique advantages
compared to the background art for silver nanoparticles. The
methods are:
[0041] green, fast, and inexpensive;
[0042] do not require polymers, surfactants, or toxic,
nondegradable reagents; and
[0043] are uncomplicated to perform and do not require
sophisticated laboratory equipment or extensively trained
technicians.
[0044] These methods synthesize nanoparticles, such as hollow
silver nanoparticles with a variety of geometries and sizes in a
very short time, for example, in less than 10 min. These methods
allow control over the size, morphology, density and surface area
of nanoparticles and the pores or hollow spaces in silver
nanoparticles and synthesize a wide variety of anisotropic silver
nanostructures, including unique 3d floral (flowerlike)
nanostructures. Some nonlimited embodiments of the invention are
described below.
[0045] A method for synthesizing silver nanoparticles comprising
chemically reducing a precursor silver compound in the presence of
a reducing agent and a capping agent for a time and under
conditions sufficient to produce silver nanoparticles. Examples of
the silver precursor compound include silver nitrate, silver
acetate, silver chloride, or other kinds of silver salts and
examples of a reducing agent include both ascorbic acid and
dextrose, or combinations of reducing agents. Some examples of a
capping agent are citric acid, sodium citrate, potassium citrate,
or other kinds of citrate salts.
[0046] Specific embodiments of this method include reducing the
precursor silver compound with ascorbic acid, with ascorbic acid
and NaOH, with dextrose, with dextrose and NaOH, with ascorbic acid
and dextrose, or with ascorbic acid and dextrose and NaOH. In other
specific embodiments of this method the capping agent will be
trisodium citrate, or the capping agent and/or morphology
controlling agent will be only dextrose, only dextrose and citrate.
A capping and/or morphology controlling agent may also constitute
one of the above-mentioned agents, like citrate, in combination
with one or more polymers, for example, citrate in combination with
at least one polymer selected from the group consisting of PEG,
PVA, PMMA, PEI, CMC and chitosan.
[0047] In conjunction with this method the precursor silver
compound, reducing agent and capping and/or morphology controlling
agent may be dissolved in water or in another aqueous solution. In
an alternative embodiment, the precursor silver compound, reducing
agent and capping agent are dissolved in an organic solvent or in a
mixture of water and an organic solvent.
[0048] One of skill in the art may select appropriate physical
conditions, such as temperature and pressure for performing this
method. For example, the method may be conducted at a temperature
below room temperature, at room temperature, or at temperature
above room temperature or by gently heating the precursor silver
compound and reducing agent. The method can performed at standard
atmospheric pressure or at a pressure above or below standard
atmospheric pressure.
[0049] The morphology of the silver nanoparticle produced by this
method can be controlled by selecting particular concentrations or
ratios of the silver precursor compound(s), reducing agent(s),
and/or capping agent(s), including polymer concentration if
present. Variable concentrations of these ingredients or physical
conditions may be provided during the reaction producing the silver
nanoparticles. The size and morphology of the nanoparticles can be
controlled by adjusting the pH, temperature, pressure and/or UV or
other kinds of radiation as well as by the other conditions
described above. For example, the size of the nanostructures
produced can be reduced by using a lower concentration of silver
compound or reducing agent than that used in an otherwise identical
method that produces larger nanostructures or vice versa. The size
of the nanostructures may also be reduced by using a lower
concentration of silver compound or reducing agent and a higher
intensity of, higher wavelength of, or longer exposure time to, UV
or other kinds of irradiation than that used in an otherwise
identical method that produces larger nanostructures and vice
versa. The terms nanoparticles and microparticles comprise those
having particle sizes in the ranges of 1-1,000 nm and 1,000-10,000
nm; respectively.
[0050] Other specific embodiments of this method include one that
uses ascorbic acid as a reducing agent, citrate as a capping and/or
shape control agent and which takes place at room temperature; one
that uses ascorbic acid as a reducing agent, citrate and one or
more polymer(s) as a capping and/or shape control agents and which
takes place in the absence of UV irradiation; one that uses
ascorbic acid as a reducing agent, citrate as a capping and/or
shape control agent and which takes place in the presence of UV
irradiation.
[0051] Another embodiment of the invention is a method for
synthesizing gold, platinum or palladium (or other noble metal)
nanoparticles comprising chemically reducing a precursor gold,
platinum, palladium or other noble metal compound in the presence
of a reducing agent and a capping agent for a time and under
conditions sufficient to produce gold, platinum or palladium
nanoparticles or microparticles.
[0052] These methods produce different kinds of nanoparticles or
microparticles which are described in more detail below and by the
figures. These include nanoparticles or microparticles exhibiting a
dendritic or fractal pattern; methods that produce a solid,
lattice-like, hollow, layered and/or generally symmetrical
nanoparticle or microparticle; methods that produce a nanoparticle
or microparticle that does not exhibit a dendritic or fractal
pattern; or methods that produce a nanoparticle or microparticle
that is not solid, lattice-like, hollow, layered and/or generally
symmetrical.
[0053] The invention is also directed to embodiments represented or
characterized by particular kinds of nanostructures. These include,
but are not limited to a nanostructure or microstructure produced
by or producible by the any of the methods or various combinations
of conditions described above. Such nanostructures include a floral
or flower-like nanostructure or microstructure; a cube or cuboid
nanostructure or microstructure; a dendrimer nanostructure or
microstructure; a bipod nanostructure or microstructure; a myriad
dendrimer nanostructure or microstructure, which has soft or smooth
arms; a star-shaped nanostructure or microstructure that has soft
or smooth arms; a star-shaped nanostructure or microstructure that
has rough arms; a star-shaped nanostructure or microstructure
having soft or rough arms containing hole(s) or an empty core; a
floral or flower-like nanostructure or microstructure having soft
or smooth arms; a floral or flower-like nanostructure or
microstructure having soft or smooth arms with a hole(s) or an
empty core; a myriad dendrimers nanostructure or microstructure
having soft or smooth arms; a butterfly-like nanostructure or
microstructure having soft or smooth branched wing-like arms; a
star-like nanostructure or microstructure having soft or smooth
arms with multiple layers of arms; a star-like nanostructure or
microstructure having rough arms and multiple layers of arms; a
star-like nanostructure or microstructure having soft or smooth
branched arms and multiple layers of arms; a star-like
nanostructure or microstructure having rough, branched arms and
multiple layers of arms; a floral or flower-like nanostructure or
microstructure having soft arms and multiple layers of arms; a
floral or flower-like nanostructure or microstructure having rough
arms and multiple layers of arms; a butterfly-like nanostructure or
microstructure having soft or smooth wing-like arms and multiple
layers of arms; a butterfly-like nanostructure or microstructure
having rough, branched wing-like arms and multiple layers of arms;
a 3d floral or flower-like nanostructure or microstructure having
multiple layers of hollows, rough surface, and external channels
surrounding the nanostructure particles substantially as shown in
FIG. 1; a 3d floral or flower-like nanostructure or microstructure
having multiple layered hollow rough surfaces, large holes, highly
external channels surround the nanostructure substantially as shown
by FIG. 2; a 3d shell-like silver nanostructure or microstructure
with little hollow pores, rough surface, highly external arms
substantially as shown by FIG. 3; a 3d scaffold-like silver
nanostructure or microstructure with little hollow pores, and a
rough surface substantially as shown by FIG. 4; a 3d roll fiber
twin-like silver structures with little hollow pores, rough
surface, highly external arms substantially as shown by FIG. 5;
porous spherical-like silver nanostructures with little hollow
pores and rough surface substantially as shown by FIG. 6; porous
spherical sponge-like silver nanostructures with high
inter-connected pores and rough surface substantially as shown by
FIG. 7; 3d flower-like silver nanostructures with multi external
interacted layers of hollow, branched rough edges substantially as
shown by FIG. 8; a flower-like silver nanostructures with multi
layers of paper, hollow cores, soft surface substantially as shown
by FIG. 9; a tree-like silver nanostructures with multi-branched
edges/arms substantially as shown by FIG. 10; a flower-like silver
nanostructure with more wide internal hollow core, multi-edges/arms
substantially as shown by FIG. 11; dendrimer silver nanostructures
with highly branched arms substantially as shown by FIG. 12; flower
silver nanostructures with more internal hollow pores and rough
surface substantially as shown by FIG. 13; octahedral multi layer
silver nanostructures (FIG. 18); octahedral multi-layered
nanostructures with pores on the surface (FIG. 19); ribbed silver
nanostructures substantially as shown by FIG. 20; multi-ribbed
nanostructures with cubes decorated on the surface of the silver
structures substantially as shown by FIG. 21; octahedral structures
with cubes decorated on the surface silver structures substantially
as shown by FIG. 22; silver stars with soft, rough arms
substantially as shown by FIG. 23 or FIG. 24 produced by a method
described herein that allows control of their size and morphology;
silver flowers with soft arms substantially as shown by FIG. 25,
produced by a method described above allowing control of their size
and morphology; myriad dendrimer with branched arms substantially
as shown by FIG. 26 produced by a method that permit the control of
their size and morphology; silver butterfly-like structures with
arms/wings substantially as shown by FIG. 27 produced by a method
that allows control of their size and morphology. These
nanoparticles or microparticles can be produced by the methods
described herein as well as by equivalent methods that make the
same kinds of structures.
[0054] Other embodiments represent compositions containing or
comprising nanoparticles or microparticles according to the
invention, such as those containing one or more additional
ingredients, such as one or more pharmaceutically or diagnostically
suitable ingredients, which may be covalently or non-covalently
associated with the nanostructure, or microstructure. A size
selected or refined nanoparticle or microparticle composition may
also be produced by selectively removing nanostructures or
microstructures, based on their size or morphology, for example, as
produced by filtration, centrifugation, or sedimentation to recover
nanostructures having a particular morphology and/or size.
[0055] Other inventive embodiments include methods of using the
nano- or microstructures described herein. These include a method
for antimicrobial therapy comprising contacting a microbe or a host
cell a nano- or microparticle as described herein. A method of
diagnosis comprising processing or contacting a biological sample
with a nano- or microparticle as described herein. A method for
detecting a molecule or substance comprising contacting a sample
with a nano- or microparticle as described herein under conditions
suitable for SERS. A method for optically detecting a molecule or
substance comprising contacting a sample with a nano- or
microparticle as described herein under conditions suitable for
optical detection. A method for optically detecting a molecule or
substance comprising contacting a sample with a nano- or
microparticle as described herein under conditions suitable for
optoelectronic detection or operation. A method for biosensing
comprising incorporating a nano- or microparticle as described
herein into a device or a biosensor. A method for tissue
engineering, treatment or modification comprising contacting a
tissue with a nano- or microparticle as described herein. An
imaging method comprising contact a sample to be imaged with the a
nano- or microparticle as described herein. A method for modeling a
system, such as a biological system, comprising contacting a system
with a nano- or microparticle as described herein. A waveguide or
electronic device comprising the a nano- or microparticle as
described herein. An ink, stain, dye, pigment, primer, paint or
anticorrosive agent comprising a nanoparticle made by the methods
describe herein. A resin, plastic, rubber, putty, or glass
comprising nanoparticles made by the methods described herein. An
agent added to a product or composition to identify, tag it, track
it or to otherwise identify it or its properties comprising
nanoparticles made by the methods describe herein. A composite that
enhances thermal, electrical or magnetic conduction comprising
nanoparticles made by the methods described herein. A composition
that attenuates thermal, electrical or magnetic conduction
comprising nanoparticles made by a method according to the
invention. A bleach, surfactant, laundry detergent, soap,
antimicrobial agent, antiseptic, or cleaning agent comprising
nanoparticles made by a method according to the invention. A
filter, HEPA filter, face mask, catalyst, or detoxifying agent or
substrate comprising a nanoparticles made by a method according to
the invention. A medical device, medical equipment or medical
supplies, such as bandages, wound dressings or biological implant
such as a heart valve, comprising nanoparticles made by a method
according to the invention. A dental device, prosthetic, dental
equipment or dental supply, such as a dental metal alloy or
artificial tooth or implant, comprising nanoparticles made by a
method according to the invention. A cosmetic or dermatological
product comprising nanoparticles made by a method of according to
the invention. A microelectromechanical system (MEMS) comprising
nanoparticles made by a method according to the invention. An
explosive, primer, propellant, shell, ammunition, weapon, or other
ordnance comprising nanoparticles made by the methods described
herein. A consumer product, such as a toy, food storage container,
baby pacifier, clothing, jewelry, metal, plastic or glass
decoration comprising nanoparticles made by the methods described
herein. A method for evaluating the safety of nanoparticles made by
the methods according to the invention comprising contacting a cell
or an organism with said nanoparticles, measuring at least one
biological effect of said nanoparticles on the cell or organism in
comparison with an otherwise identical cell or organism not exposed
to said nanoparticles.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] The patent or application file contains at least one drawing
executed in color.
[0057] Gold Nanoparticle-based Assays
[0058] FIG. 1. Flow chart of one embodiment of an cationic
AuNP-based assay.
[0059] HCV virions are first lysed with RNA lysis buffer and
proteins are digested by Proteinase K. HCV RNA present in the
digested lysate is captured with silica nanoparticles conjugated to
an oligonucleotide specific for the target HCV RNA. The captured
target RNA is then washed and then eluted in a purified form. The
purified RNA is added to AuNPs in a phosphate buffer. A color
change from red to blue observed within about 2 minutes. This
change in color occurs due to alignment of the positively charged
AuNPs on the phosphate backbone of the HCV RNA. AuNPs in samples to
which no RNA is added do not aggregate and remain red.
[0060] FIG. 2. UV-Vis spectrum of the positive AuNPs. The spectrum
shows a .lamda..sub.max at 531 nm characteristic of spherical
AuNPs.
[0061] FIG. 3. (A) SEM of the prepared AuNPs. (B) The particles
have an average diameter of about 30 nm.
[0062] FIG. 4. Analysis of clinical specimens using AuNP-based
assay. In the absence of HCV RNA, the AuNPs are positively charged
and are separated from each other by repulsion and the solution is
red in color. On the other hand, in samples containing HCV RNA the
AuNPs align on the phosphate backbone of the HCV RNA changing the
color of these HCV RNA-positive samples from red to blue.
[0063] FIG. 5. (A) SEM of the prepared silica nanoparticles. (B)
Diameter calculation of the prepared silica nanoparticles.
[0064] FIG. 6. (A) SEM of the prepared magnetic nanoparticles. (B)
Diameter calculation of the magnetic nanoparticles. (C) FT-IR of
magnetic nanoparticles functionalized with amino propyl tri-ethoxy
silane (APES), The band at 583 cm.sup.-1 corresponds to the Fe--O
bond, the one at 1050 cm.sup.-1 and 1380 cm.sup.-1 corresponds to
the vibrations of SiOCH.sub.2 structure and Si--CH.sub.2 scissoring
vibrations respectively, while the N--H bending mode and stretching
vibrations of the free amino groups are appeared at bands 1625
cm.sup.-1 and 3436 cm.sup.-1 respectively. Also, the anchored
propyl group of the APTES is present at band 2923 cm.sup.-1
[0065] FIG. 7 illustrates the mechanism of how cationic AuNPs
aggregate along a strand of nucleic acid.
Anisotropic Silver Nanoparticles
[0066] FIG. 8 shows SEM photographs of 3d flower-like silver
structures with hollow multi layers (3-15 layers), surface
roughness (10-200 nm), external channels (1-4) surrounding the
particles.
[0067] FIG. 9. SEM of 3d flower-like silver structures with more
multi layer (16-30 layers) of hollow, surface roughness (250-400
nm), with larger holes (50-200 nm), highly external channels (5-10)
surrounding the particles.
[0068] FIG. 10. 3D scaffold fibers like (B), flakes (C), and
cluster (D) Silver structures before centrifugation (A). 3D
scaffold like with hollow layer (10-50 nm in width and 100-500 nm
length) and rough surface (150-500 nm). Silver flakes like (10-50
nm in width and 20-200 nm length.
[0069] FIG. 11. SEM of 3d scaffold-like silver structures with
hollow pores (20-80 nm in width, 500-900 nm length), rougher
surface (400-500 nm).
[0070] FIG. 12. SEM of 3d roll fiber twin-like silver structures
with little hollow pores (50-200 nm), rougher surface (500-700 nm),
highly external arms (30-40 arms) surrounding the particles.
[0071] FIG. 13. SEM of porous spherical-like silver structures with
little hollow pores (100-200 nm width, 700-900 nm length), rougher
surface 0.9-1.5 .mu.m.
[0072] FIG. 14 SEM of porous spherical sponge-like silver
structures with high inter connected pores (150-300 nm), more rough
surface (1.6-2.0 .mu.m).
[0073] FIG. 15 SEM of 3d flower-like silver structures with multi
external interacted layers (15-40 layers) of hollow, branched (3-9)
rough edges.
[0074] FIG. 16. SEM of flower-like silver structures with multi
layer of paper (5-30 layers), hollow-like cores (10-80 nm), soft
surface.
[0075] FIG. 17. SEM of tree-like silver structures with multi
branched edges like arms (20-50 arms).
[0076] FIG. 18. SEM of flower-like silver structures with wide
internal hollow-like core (500-700 nm wide), multi-edge like arms
(3-12 arms).
[0077] FIG. 19. SEM of dendrimer silver structures with highly
branched arms (5-40 arms).
[0078] FIG. 20. SEM of unique flower-silver structures with more
internal holes (150-500 nm), rough surface (1.5-3.0 .mu.m).
[0079] FIG. 21. SEM of cube silver structures with soft surface and
ranged edges.
[0080] FIG. 22. SEM of pyramidal silver structures.
[0081] FIG. 23. SEM of bibode silver structures.
[0082] FIG. 24. SEM of triangular silver structures.
[0083] FIG. 25. SEM of octahedral multi layer (2-7 layers) silver
structures.
[0084] FIG. 26. SEM of octahedral multi layer (3-5 layers), with
pores (1-5 pores) on the surface.
[0085] FIG. 27. SEM of ribbed silver structures were
synthesized.
[0086] FIG. 28. SEM of multi-ribbed (5-15) with cubes decorated on
the surface silver structures.
[0087] FIG. 29. SEM of octahedral with cube decorated on the
surface silver structures.
[0088] FIG. 30. SEM of silver stars prepared by using varying
amounts of 0.5 ml of 0.0001-2.0 M TSC; 50, 100, 200, 300, 400, 500,
600, 700, or 800 .mu.L of 0.001-0.004 mM AgNO.sub.3; and 100 .mu.L
of 0.3-3.0 M ascorbic acid.
[0089] FIG. 31. SEM of silver stars prepared by using varying
amounts (0, 300, 400, 500, 600, 700, 800, 1100 .mu.L) of 0.0001-2.0
M TSC; 0.3 mL of 0.001-0.1 M AgNO.sub.3; and 100 .mu.L of 0.3-3.0 M
ascorbic acid.
[0090] FIG. 32. (A-C) SEM of silver flowers prepared by using 50,
100, 150, 200, 250, or 300 .mu.L of 0.3-3.0 M ascorbic acid, 300
.mu.L 0.001-0.004 mM AgNO.sub.3; and 0.5 mL of 0.0001-2.0 M
TSC.
[0091] FIG. 33. SEM of silver Myriad Dendrimer prepared by using of
300, 200, or 100 .mu.L of 0.0001-2.0 M TSC; and 300, 200, or 100
.mu.L of 0.001-0.1 mM AgNO.sub.3; and 100 .mu.L of 0.3-3.0 M
ascorbic acid.
[0092] FIG. 34. SEM of silver butterfly prepared by using of 500
.mu.L of 0.0001-2.0 M TSC; 300, 200, or 100 .mu.L of 0.001-0.1 mM
AgNO.sub.3; and 80, 100 and/or 150 .mu.L of 0.3-3.0 M ascorbic
acid.
[0093] FIG. 35. SEM of silver stars prepared by using 0.3 mL of
polymer solution (PEG, PVA, or PEI), subjected to 18 W ultraviolet
light at wave length (430 nm) for 30 min, using 500 .mu.L of
0.0001-2.0 M TSC; 300, 200, 100 .mu.L of 0.001-0.1 mM AgNO.sub.3;
and 80, 100 and/or 150 .mu.l, of 0.3-3.0 ascorbic acid.
[0094] FIG. 36. SEM of silver stars prepared by using of 0.3 mL of
polymer solution (PEG, PVA, or PEI) without using UV irradiation,
using 500 .mu.L of 0.0001-2.0 M TSC with using of 300, 200, or 100
.mu.L of 0.001-0.1 mM AgNO.sub.3; and 80, 100, and/or 150 .mu.L) of
0.3-3.0 M ascorbic acid.
[0095] FIG. 37. Silver spherical nanoparticles by using of 0.4,
0.6, and 0.8 ml of dextrose respectively (A-C).
[0096] FIG. 38. Silver stars nanostructures that synthesized by
using of 0.3 ml PVA with UV (A) and without UV, while C and D were
synthesized by using of 0.3 ml PMMA without and with UV
respectively.
[0097] FIGS. 39-67 show the UV/Vis/NIR extinction spectra of the
silver structures.
[0098] FIG. 68. Diagram showing Synthesis of liposome silver
nanoparticles (LAgNPs).
[0099] FIG. 69. (A) TEM of the AgNPs and (B) UV spectrum for AgNPs
and photo of prepared AgNPs.
[0100] FIG. 70. (A-C) SEM of the nanoliposomes prepared using 1:1
(A), 1:2 (B), and 1:3 molar ratios of PC:Cho. TEM of LAgNPs
prepared with 1 molar ratio of AgNPs and 1:1 (D), 1:2 (E), and 1:3
(F) molar ratios of PC:Cho; respectively.
[0101] FIG. 71. Cumulative release of AgNPs over time from LAgNPs
prepared with 1:1, 1:2, or 1:3 molar ratios of PC:Cho and 1 molar
ratio of AgNPs. Diffusion of free AgNPs through cellulose membrane
was used as a control.
[0102] FIG. 72. Antimicrobial effects of nanoliposomes (A), free
AgNPs (B), and LAgNPs (C) against four different bacterial strains.
In all experiments, bacterial growth was determined by reading
absorbance at 600 nm after 24 h of treatment. Nanoliposomes used
were prepared using 1PC:3 Cho.
PART 1: DETAILED DESCRIPTION OF THE INVENTION
[0103] Gold Nanoparticle-Based Assays.
[0104] The inventors have developed for the first time an
AuNP-based colorimetric method (solution phase) utilizing
non-functionalized positively charged AuNPs for the direct
detection of unamplified RNA and/or DNA molecules extracted from
clinical specimens. This method can be applied to identify
biomarkers of diseases such as nucleic acids and/or proteins from
eukaryotic or prokaryotic sources particularly that of pathogens.
In the examples shown below, Hepatitis C Virus (HCV) RNA was used
as a model for testing the efficiency and specificity of the
developed assay, which was used for the direct and specific
detection of unamplified HCV RNA extracted from clinical
samples.
[0105] HCV is a major global health problem as it infects about 200
million individuals worldwide, with 3-4 million newly infected
annually [7]. Chronic Hepatitis C develops in about 70-90% of cases
and about 5-20% and 1-5% of chronically infected patients develop
cirrhosis and hepatocellular carcinoma (HCC), respectively [8]. As
illustrated in FIG. 1, this method can employ a selective
extraction method that captures the nucleic acid of interest from
clinical specimens and then colorimetrically identify the captured
nucleic acid. This method is simple, rapid, highly sensitive,
specific and cost-effective.
[0106] HCV RNA was extracted using a commercially available viral
RNA extraction kit (Promega). Extracted HCV RNA was then added to
the positively-charged AuNPs. In case of positive samples, the
positively-charged AuNPs bind to the negatively-charged phosphate
groups on the HCV RNA target via electrostatic attraction between
the negatively charged RNA phosphate backbone and the positively
charged CTAB capping on AuNPs leading to Plasmon-Plasmon
interaction and a change in the solution colour from red to
blue.sup.2,13. In the absence of HCV RNA (absence of any nucleic
acid), the AuNPs are far from each other (repulsion between
positively-charged particles) and thus the solution colour remains
red (FIG. 4). Since the developed assay utilizes positively-charged
AuNPs instead of negatively charged ones for HCV RNA detection, the
test is more direct and simpler requiring minimal optimization. It
is important to note that the red colour is stable even when left
undisturbed for several days. Thus, the developed assay can simply
and rapidly (about 5 min) determine any nucleic acid (RNA and/or
DNA single or double stranded) in the solution. The reported
sensitivity and specificity is 91%. and 94.8%, respectively.
[0107] The presence of nucleic acid in the sample, leads to
alignment of the positively charged AuNPs on the phosphate backbone
of the nucleic acid, the presence of phosphate buffer in the assay
increases the aggregation capability of the AuNPs (for only the
positive samples) by binding of the phosphate ions to the aligned
AuNPs and then another row of AuNPs attached to the other part of
the phosphate ions which will be aligned on another phosphate
backbone of another RNA molecule (FIG. 7). So, aggregation occurs
by first alignment of AuNPs on phosphate backbone of one nucleic
acid, which then binds to phosphate ions and the latter binds to
another AuNPs which are aligned on another nucleic acid phosphate
backbone. Therefore, at least 5 layers are formed in the positive
samples and leads to AuNPs aggregation, the layers are:
1stRNA/AuNPs/Phosphate ions/AuNPs/2ndRNA. These layers will result
in forming a network of nucleic acid/AuNPs which lead to
aggregation of gold nanoparticles and change of colour from red to
blue.
[0108] To increase specificity and sensitivity of the assay, HCV
RNA specific probe conjugated to silica and/or magnetic
nanoparticles have been used for viral RNA capture after lysis the
virion and precipitation of the proteins in the sample so a simple,
rapid, specific and direct magnetic/silica-probe based extraction
method has been developed and when coupled with the AuNP based
colorimetric assay a sensitivity and specificity of 92.6% and
96.5%, respectively, was obtained.
[0109] The invention is not limited to a particular method for
synthesizing cationic (positively charged) gold nanoparticles
(AuNPs) or silver nanoparticles (AgNPs). Various methods may be
used. General synthesis methods for producing positively charged
gold nanoparticles are based mainly on the reduction of hydrogen
tetrachloroaurate trihydrate (HAuCl.sub.4) using sodium borohydride
as a reducing agent, in the presence of the capping agent. Changing
concentration of the different reagents, reaction time and pH will
determine the final size and shape of the prepared nanoparticles.
The common used capping agents are:
[0110] (i) cetyl trimethyl ammonium bromide (CTAB), see Narayanan
R, Lipert R. J, Porter M D. Cetyltrimethylammonium bromide-modified
spherical and cube-like gold nanoparticles as extrinsic Raman
labels in surface-enhanced Raman spectroscopy based heterogeneous
immunoassays. Analytical chemistry. 2008, 80(6): 2265-71; and
Wenlong Cheng, Shaojun Dong, Erkang Wang. Synthesis and self
assembly of cetyltrimethylammonium bromide capped gold
nanoparticles. Langmuir. 2003, 19(22): 9434-39 (both of which are
incorporated by reference);
[0111] (ii) cysteamine, see Kim J W, Kim J H, Chung S J, Chung B H.
An operationally simple colorimetric assay of hyaluronidase
activity using cationic gold nanoparticles. Analyst. 2009 July;
134(7):1291-3; or T. Niidome et al. Preparation of primary
amine-modified gold nanoparticles and their transfection ability
into cultivated cells. Chem. Commun. 2004, (17):1978-1979 (both of
which are incorporated by reference); and
[0112] (iii) lysine, see P R. Selvakannan et al. Capping of gold
nanoparticles by the amino acid lysine renders them water
dispersible. Langmuir. 2003, 19(8): 3345-49 (which is incorporated
by reference).
[0113] Methods used for synthesis of positively charged silver
nanoparticles (AgNPs), are similar to those used for the gold
nanoparticles [9], and based on the reduction of silver salt in the
presence of capping agents as CTAB [10, 11], polyethylimine (PEI)
[12] and p-benzoquinone [13]. Silver nanoparticles based assays are
based on change of color of the silver nanoparticles solution from
yellow to colorless and/or formation of white precipitate based on
the degree of aggregation.
[0114] The present invention is described below based on some
specific examples which pertain to some specific embodiments of the
invention. However, the invention is not limited to what is
described in these examples.
EXAMPLES
Example 1
Detection of HCV RNA in Serum Samples
Synthesis of Positively-Charged AuNPs
[0115] Positively charged spherical particles were synthesized as
previously described in [14]. Briefly, the seed solution was
prepared by reducing HAuCl.sub.4 (2.5 ml of 0.001 M HAuCl.sub.4),
in presence of CTAB (7.5 ml of 0.2 M), with ice-cold NaBH.sub.4
(600 .mu.L; 0.01 M). The vials were then shaken vigorously (about 2
min) to produce brown seed suspensions. The seed (80 .mu.l) was
then added to the centre of a solution containing HAuCl.sub.4 (50
ml of 0.001 M HAuCl.sub.4), CTAB/BDAC mixture (50 mL containing 0.2
M of CTAB plus 0.25M of BDAC), AgNO3 (1.5 mL of 0.004 M AgNO.sub.3)
and ascorbic acid (700 .mu.l of 0.0788 M). The mixture was left
undisturbed for about 24 h.
[0116] Characterization of Synthesized AuNPs
[0117] The absorbance spectrum and concentration of the positively
charged particles were determined using UV spectrophotometer
(Jenway 6800) as previously reported in[15]. The shape and size of
the produced positively charged particles were analyzed using field
emission scanning electron microscopy (SEM; Model: Leo Supra 55;
US). For the SEM analysis, 5 .mu.l of the synthesized AuNPs were
placed on silicon wafer and allowed to air dry before examination.
UV-Vis spectrum was performed for the prepared AuNPs. The spectrum
shows a .lamda..sub.max at 531 nm characteristic of spherical AuNPs
(FIG. 2) [14]. Scanning Electron Microscope (SEM) image (FIG. 3A)
was analyzed by (Image J 1.4 software Wayne Rasband, National
Institutes of Health, USA. http://_rsb.info.nih.gov/ij/java
1.6.0.sub.--05). The particles had an average diameter of about 30
nm and spherical in shape (FIG. 3B).
[0118] Isolation of Nucleic Acid from Serum
[0119] Serum Sample Collection.
[0120] Serum samples were collected from healthy volunteers (n=38)
and from chronic HCV patients (n=48). Rapid HCV test was performed
on all the samples. All positive samples had elevated ALT and AST
levels. All samples were negative for hepatitis B surface antigen
and hepatitis B antibody.
[0121] Extraction of HCV RNA.
[0122] Extraction of HCV RNA from serum samples was performed using
SV total RNA isolation System (Promega; Cat. No. Z3100) according
to the modified manufacturer's protocol for HCV RNA isolation
[16].
[0123] Real-Time RT-PCR.
[0124] Real-time RT-PCR was performed using AgPath ID One Step
RT-PCR kit (cat #AM1005; Ambion) [17] according to manufacturers
protocol. To 16.5 .mu.l master mix, 8.5 .mu.l of the extracted HCV
RNA was added and amplification was performed using Stratagene
(Mx3005P) under the following cycling conditions: 1 cycle of
45.degree. C. for 10 min, 1 cycle of 95.degree. C. for 10 min
followed by 45 cycles of 95.degree. C. for 15 s and 60.degree. C.
for 45 s.
[0125] Colorimetric AuNP-Based Assay
[0126] To 5 .mu.L of the extracted HCV RNA, 5 .mu.L of 2M Phosphate
buffer was added followed by 30 .mu.L of the positively charged
AuNPs. The sample was mixed by pipetting and the color of the
solution was observed within 5 minutes.
Example 2
Silica Nanoparticle Capturing Method
[0127] Colloidal Silica Nanoparticles: Synthesis and
Functionalization
[0128] HCV RNA was extracted using colloidal silica nanoparticles
conjugated to an oligonucleotide specific to HCV RNA. Initially,
200 nm colloidal silica nanoparticles were synthesized with a
modified stober method (modification was done in our lab). Briefly,
in a beaker mix absolute ethanol, deionized water, concentrated
ammonia and tetraethyl orthosilicate (TEOS), and stir at room
temperature for about 1 hour. Then, the formed colloidal solution
was centrifuged at 4000 rpm for 10 minutes, and the supernatant was
discarded and the pellet was washed with absolute ethanol. This
washing step was repeated for about 4 times or until no ammonia
odor in the solution. The pellet was then dispersed in absolute
ethanol and sonicated for about 5 minutes to remove any aggregates.
The produced silica nanoparticles were examined using scanning
electron microscope (SEM), to get the morphology and the diameter
of the prepared silica nanoparticles (FIG. 5).
[0129] The prepared silica nanoparticles were functionalized with
amino propyl trimethoxy silane (APMS) to introduce amino groups on
the surface of the silica nanoparticles. Briefly, 1 ml of APMS was
added to 20 ml of the prepared colloidal silica nanoparticles and
stirred at room temperature for at least 2 hours, then the solution
was centrifuged at 4,000 rpm for 10 minutes, and the supernatant
was discarded and the pellet resuspended in phosphate buffer saline
(PBS 1.times.). The number of silica nanoparticles per ml was
calculated as previously described [18]. Briefly, one ml of the
prepared silica colloidal solution was taken centrifuged, and the
supernatant was discarded, then the pellet was dried till complete
dryness. The dried pellet was then weighted in milligrams and from
the volume taken (1 ml) and the weight obtained, concentration of
the colloidal solution has been calculated which is 12 mg/ml. From
FIG. 6 the diameter of the silica nanoparticles was measured to be
about 150 nm. The weight of one particle equals volume of the
particle * specific gravity (2.3), the volume equals 4/3 .pi. r3,
and the particles count equals concentration (12 mg/ml)/weight of
one particle equals 2.95125E+12 silica nanoparticles/ml.
Synthesis of Silica Probe
[0130] A heterobifunctional cross linker (3-maleimidobenzoic acid
N-hydroxyl succinimide, MBS) was used to prepare an HCV specific
probe conjugated to silica nanoparticles. This linker has NHS ester
at one end which reacts with primary amine groups to form stable
amide bond; the other end has maleimide group which reacts with
sulfhydryl groups. The cross linker binds to the amine
functionalized silica nanoparticles through the NHS ester and to a
thiolated HCV specific probe through the maleimide group. The thiol
labeled probe was prepared as previously described [19, 20]. 10 mg
of MBS dissolved in 1 mL dimethyl formamide plus 2.5 ml PBS and 2.5
ml of amino functionalized silica nanoparticles and mix at room
temperature for at least 2 hours, and then purified by
centrifugation, the thiol modified probe was added to the MBS
conjugated silica nanoparticles and incubate at room temperature
for at least 2 hours. The number of probes per one silica
nanoparticle was calculated by first multiplying the number of
moles of the probe by Avogadro's number, and then dividing the
number of probes calculated by the silica nanoparticles count, and
it was about 500 probes per one silica nanoparticle.
[0131] Extraction of HCV RNA from Clinical Samples Using the
Prepared Silica Probes
[0132] To 200 .mu.l of patient sera, 200 .mu.l of lysis buffer
(Promega SV viral RNA) was added. After mixing by inversion, 50
.mu.l Proteinase K was added and left to incubate for 10 min. The
mixture was heated to 95.degree. C. in a heat block for 2 min then
50 .mu.l silica-probes was added and the reaction mixed for 1 hr.
The mixture was centrifuged at 3000 RPM for 3 min and the pellet
was washed twice with nuclease-free water. The HCV RNA was then
eluted by heating at 95.degree. C. for 5 min. The mixture was
centrifuged and the supernatant contained the eluted RNA was
separated. The extracted RNA was tested using both Real-time RT-PCR
and the developed colorimetric AuNP-based assay.
[0133] Comparison Between Colorimetric AuNP-Based Assay and
Real-Time RT-PCR
[0134] HCV RNA extracted by the Promega kit or by the silica probe
developed by the inventors was detected and quantified using
Real-Time RT-PCR as described above. The assay developed by the
inventors using positively charged AuNPs was performed on the
samples extracted using the silica probe to detect HCV RNA. The
color of the AuNPs colloidal solution of the negative samples
remained red in color which indicates no nucleic acid was present
in the sample.
[0135] On the other hand, the presence of HCV RNA in the positive
samples lead to aggregation of the AuNPs and the color changed from
red to blue (FIG. 4), the intensity of the blue color in positive
samples was compatible with the viral load as quantified by
Real-Time PCR. Of the 68 HCV positive samples, 63 samples gave blue
color which indicates the presence of the HCV RNA, while no change
in color occurred in 5 samples (False negative).
[0136] On the other hand, 56 out of 58 negative samples gave red
color which indicates the absence of HCV RNA in addition to any
other nucleic acid (high purity of the sample), while one sample
only gave blue color due to AuNPs aggregation (False Positive).
These results show that the cationic AuNP based assay has
specificity of 96.5%, and a sensitivity of 92.6%.
Example 3
Magnetic Nanoparticle Capturing Method
Iron Oxide Magnetic Nanoparticles: Synthesis and
Functionalization
[0137] HCV RNA was extracted using homemade magnetic nanoparticles
conjugated to an oligonucleotide specific to HCV RNA. First, 90 nm
magnetic nanoparticles were synthesized as described elsewhere
[21]. Typically iron (II) chloride and iron (III) chloride (1:2)
were dissolved in nanopure water at the concentration of 0.25 M
iron ions and chemically precipitated at room temperature
(25.degree. C.) by adding 1 M NaOH at a constant of pH 10. The
precipitates were heated at 80.degree. C. for 35 min under
continuous mixing and washed four times in water and several times
in ethanol. During washing, the magnetic nanoparticles were
separated from the supernatant using a magnet, and the particles
were finally dried in a vacuum oven at 70.degree. C.
[0138] Amino functionalization of the prepared magnetic
nanoparticles was done by amino propyl Trimethoxy silane (APMS) as
described elsewhere[22]. Briefly, magnetic nanoparticles (1 g) were
washed with 99.5% methanol and twice with Nanopure water and soaked
in 10 mL of 3 mM APTMS solution in a toluene/methanol (1:1 v/v)
mix. The suspension was then transferred into a three-necked flask
with a water-cooled condenser and temperature controller with a
nitrogen gas flow at 80.degree. C. for 20 h under vigorous
stirring. Silanization was found to occur at the surfaces of the
particles bearing hydroxyl groups, which in the presence of an
organic solvent results in the formation of an APTMS coating with a
large density of amines. The particles were recovered by applying
an external magnetic field after the silanization process and
washed three times with methanol and dried at 50.degree. C. in a
vacuum oven.
[0139] Characterization of Magnetic Nanoparticles
[0140] The prepared magnetic nanoparticles were characterized by
SEM for size and particles distribution determination. Moreover,
Fourier transform infrared spectroscopy (FT-IR) was used to record
the IR spectra of the samples using potassium bromide (KBr) pellet
technique.
[0141] Conjugation of HCV specific probe to the amino
functionalized magnetic nanoparticles
[0142] To prepare the magnetic nanoparticles conjugated to HCV RNA
specific probe, heterobifunctional cross linker (3-maleimidobenzoic
acid N-hydroxyl succinimide, MBS) was used that has NHS ester at
one end that reacts with primary amine groups forming stable amide
bond, and the other end has maleimide group that reacts with
sulfhydryl groups forming stable thioether linkage.
Functionalization procedures were done as following: Firstly, the
disulfide labeled probe was prepared as previously described [19,
20]. Briefly, disulfide cleavage of the probe was done by
lyophilization of 10 nmol of the probe and then resuspended in 100
ul of 0.1 M dithiothrietol (DTT) prepared in disulfide cleavage
buffer (170 mM phosphate buffer, pH=8). The solution was wrapped in
foil and let to stand at room temperature for 2-3 h with occasional
vortexing. Desalting of the freshly cleaved probe was done using
Nap-5 column (illustra NAP-5 (GE Healthcare) according to the
manufacturer's instructions. UV-visible spectrophotometer was used
to determine the purified probe concentration. Secondly, the amine
functionalized magnetic nanoparticles were washed with Dimethyl
sulfoxide (DMSO) for 2 times, the wash discarded, then MBS cross
linker dissolved in DMSO was added to the washed magnetic
nanoparticles The mixture was allowed to mix on a roller shaker for
about 1 hour at room temperature. Then, the nanoparticles were
washed with DMSO twice followed by coupling buffer (100 mM
phosphate buffer and 0.2 M sodium chloride, pH=7) twice. Then, the
particles were resuspended again in coupling buffer and the cleaved
probe were added to the suspended particles and allowed to react on
a roller shaker overnight. Finally, the supernatant was removed and
the magnetic nanoparticles functionalized with HCV RNA specific
probe were resuspended in storage buffer (10 mM phosphate buffer,
0.1 M sodium chloride, pH=7.4).
[0143] Extraction of HCV RNA from Clinical Samples Using the
Prepared Probes
[0144] To employ specificity and selectivity of the developed AuNP
assay for a specific viral RNA/DNA, HCV RNA specific probe
conjugated to magnetic nanoparticles was used for HCV RNA capture
after virion lysis and digestion of the proteins in patient sera.
In a 1.5 ml microcentrifuge tube, 100 ul of the magnetic
nanoparticles conjugated to HCV specific probe was taken and washed
twice with the assay buffer (10 mM phosphate buffer, 150 mM sodium
chloride, pH=7.4) and then the modified magnetic nanoparticles were
resuspended in 50 ul assay buffer.
[0145] In another 1.5 ml microcentrifuge tube 200 .mu.l of patient
sera was added to 200 .mu.l of lysis buffer to break down the viral
envelope. After mixing by inversion, 50 .mu.l Proteinase K was
added to digest the serum proteins and left to incubate for 10 min.
Then, the mixture was centrifuged for 10 minutes at 14,000 rpm and
the supernatant was taken and mixed with 300 ul of iso-propanol.
Then, the resuspended magnetic nanoparticles were added to the
previous mixture and heated at 90.degree. C. for 2 minutes to
denaturate the target RNA. The mixture was shaken at a temperature
.about.15.degree. C. below the melting point of the conjugated
probe for 45 minutes. Then, the tubes were placed on magnet until
all solutions were clear and the supernatant were removed. Then,
the particles were washed twice with washing buffer (60 mM
potassium acetate, 10 mM Tris-HCl, 60% ethanol, pH=7.5).
Supernatant was removed between each wash with the help of magnet.
Elution was done by adding 50 .mu.l DEPEC-Water, and heated at
95.degree. C. for 2 minutes. The tubes were placed on magnet until
all solutions were clear and the eluted HCV RNA was transferred to
new RNase free tube. After HCV RNA extraction the colorimetric gold
nanoparticles based assay was performed as described before.
[0146] HCV RNA Capturing
[0147] One hundred twenty six samples were used in this study: 68
samples from HCV positive subjects and 58 samples from healthy
individuals. Each sample was divided and each part was subjected to
extraction by one of two different methods: (i) by use of an SV
total RNA isolation kit or (ii) by use of the developed silica
and/or magnetic probe. The presence of the HCV RNA in the positive
samples and the absence of HCV RNA in the negative samples
extracted by the silica probe were confirmed with Real-Time PCR and
compared with the samples extracted by the promega kit. The
concordance between RNA extraction by the promega kit and the RNA
extraction by the silica and/or magnetic probe was 100%, (which
means that the developed silica and/or magnetic probe could be used
alone for HCV RNA extraction without the need of any other
commercial extraction kit. Moreover, the same principle can be used
in extraction and purification of any other nucleic acid and/or
protein by simply replace the HCV RNA specific probe with the other
target specific molecule (e.g. antibodies, lectins, probes . . .
etc), therefore the developed HCV RNA extraction method by the
silica probe could be expanded to be used in many other targets.
The main aim for capturing the HCV RNA capturing is to increase the
purity of the sample from any other nucleic acids (DNA or RNA) that
may interfere with the assay results and thus allowing an increase
in assay specificity.
[0148] Other Applications
[0149] The inventors have developed a novel colorimetric
solution-phase cationic AuNP-based assay for the direct detection
of unamplified RNA/DNA. This method has been exemplified using HCV
RNA extracted from clinical specimens as a model nucleic acid. The
developed assay is not complex because it simply requires adding
cationic AuNPs solution to the target molecule (e.g., extracted RNA
in presence of phosphate buffer, rapid (about 5 min), sensitive,
cost-effective, and can be easily automated. Conveniently, the
developed assay can be used as a platform for the detection of any
RNA and/or DNA in solution form. In other words, if a specific
nucleic acid extraction method is available for any nucleic acid to
be determined; this assay provides an easy, simple and rapid way
for its analysis.
[0150] This method provides a foundation for development of other
silica-probe based extraction methods for extraction of specific
kinds of nucleic acids, such as HCV RNA.
[0151] This assay can be practiced quantitatively since the
intensity of the blue colour produced by aggregation of gold and/or
silver nanoparticles reflects the number of RNA/DNA molecules
present in the extracted sample and corresponds to factors such as
bacterial or viral load. Based on the disclosure above one of skill
in the art could determine the qualitative or quantitative
detection limit of an assay for a particular kind of nucleic acid,
its linear range, accuracy and precision.
REFERENCES
Description of Gold Nanoparticle-Based Assay
[0152] 1. Radwan, S. H. and H. M. Azzazy, Gold nanoparticles for
molecular diagnostics. Expert Rev Mol Diagn, 2009. 9(5): 511-24.
[0153] 2. Cao R, et al., Naked-eye sensitive detection of nuclease
activity using positively-charged gold nanoparticles as
colorimetric probes. Chem. Commun., 2011. 47(45): 12301-12303.
[0154] 3. Kim J W, et al., An operationally simple colorimetric
assay of hyaluronidase activity using cationic gold nanoparticles.
Analyst, 2009. 134(7): 1291-1293. [0155] 4. Ma Z, et al., Optical
DNA detection based on gold nanorods aggregation. Anal chem Acta,
2010. 673(2): 179-184. [0156] 5. Sun, Y., et al., Microarray gene
expression analysis free of reverse transcription and dye labeling.
Anal Biochem, 2005. 345(2): 312-9. [0157] 6. Hsiao C R and C. C H,
Characterization of DNA chips by nanogold staining. Anal Chem,
2009. 389(2): 118-123. [0158] 7. WHO
http://www.who.int/vaccine_research/diseases/hepatitis_c/en/[last
accessed on Jul. 22, 2010]. [0159] 8. Gourley, P. L., Brief
overview of BioMicroNano technologies. Biotechnol Prog., 2005. 21
(1): 2-10. [0160] 9. Tolaymat, T. M., et al., An evidence-based
environmental perspective of manufactured silver nanoparticle in
syntheses and applications: A systematic review and critical
appraisal of peer-reviewed scientific papers. science of the total
environment, 2009. 408: 999-1006. [0161] 10. Sui, Z., et al.,
Capping effect of CTAB on positively charged Ag nanoparticles.
Physica E.: Low-dimensional systems and nanostructures, 2006.
33(2): 308-314. [0162] 11. Khan, Z., et al., Preparation and
characterization of silver nanoparticles by chemical reduction
method. Colloids and Surfaces B: Biointerfaces, 2011. 82: 513-517.
[0163] 12. Siliu Tan, et al., Synthesis of positively charged
silver nanoparticles via photoreduction of AgNO3 in branched
Polyethyleneimine/HEPES solutions. langmuir, 2007. 23(19):
9836-9843. [0164] 13. Kima, J., S. W. Kang, and Y. S. Kang,
Partially positively charged silver nanoparticles prepared by
p-benzoquinone. Colloids and SurfacesA: Physicochem. Eng. Aspects,
2008. 320: 189-192. [0165] 14. Huang X: Gold Nanoparticles Used in
Cancer Cell Diagnostics, Selective Photothermal Therapy and
Catalysis of NADH Oxidation Reaction. Laser Dynamic Laboratory,
School of Chemistry and Biochemistry Doctor of philosphoy, 234
pages (2006). [0166] 15. Jain, P. K., et al., Calculated Absorption
and Scattering Properties of Gold Nanoparticles of Different Size,
Shape, and Composition: Applications in Biological Imaging and
Biomedicine. J. Phys. Chem. B, 2006. 110(14): 7238-7248. [0167] 16.
Link, S, and M. A. El-Sayed, Spectral Properties and Relaxation
Dynamics of Surface Plasmon Electronic Oscillations in Gold and
Silver Nanodots and Nanorods. J. Phys. Chem. B, 1999. 103(40):
8410-8426. [0168] 17. Wagner, V. et al., The emerging nanomedicine
landscape. Nat Biotechnol, 2006. 24(10): 1211-7. [0169] 18.
Nakamura, M., M. Shono, and K. Ishimura, Synthesis,
characterization, and biological applications of multifluorescent
silica nanoparticles. Anal Chem, 2007. 79(17): 6507-14. [0170] 19.
Rosi, N. L., et al., Oligonucleotide-modified gold nanoparticles
for intracellular gene regulation. Science, 2006. 312: 1027-1030.
[0171] 20. Haley D Hill and C. A. Mirkin, The bio-barcode assay for
the detection of protein and nucleic acid targets using DTT-induced
ligand exchange. Nature Protocols 2006. 1: 324-335. [0172] 21.
Kouassi, G. K., J. Irudayaraj, and G. McCarty, Activity of glucose
oxidase functionalized onto magnetic nanoparticles. Biomagn. Res.
Technol., 2005. 3(1): 1. [0173] 22. Kouassi, G. K. and J.
Irudayaraj, Magnetic and gold-coated magnetic nanoparticles as a
DNA sensor. Anal. Chem, 2006. 78(10): 3234-41.
PART 2: DETAILED DESCRIPTION OF THE INVENTION
Anisotropic Silver Nanoparticles
[0174] The synthesis of anisotropic silver nanoparticles is a
time-consuming process and involves the use of expensive toxic
chemicals and specialized laboratory equipment. The presence of
toxic chemicals in the prepared anisotropic silver nanostructures
hindered their medical application. The inventors have developed a
fast and inexpensive method for the synthesis of three dimensional
hollow flower-like silver nanostructures without the use of toxic
chemicals. In this method, silver nitrate was reduced using
dextrose in presence of trisodium
citrate as a capping agent. Sodium hydroxide was added to enhance
reduction efficacy of dextrose and reduce time of synthesis. The
effects of all four agents on the shape and size of silver
nanostructures were investigated. Robust hollow flower-like silver
nanostructures were successfully synthesized and ranged in size
from 0.2 .mu.m to 5.0 .mu.m with surface area between 25-240 m2/g.
Changing the concentration of silver nitrate, dextrose, sodium
hydroxide, and trisodium citrate affected the size and shape of the
synthesized structures, while changing temperature had no effect.
The disclosed method is simple, safe, and allows controlled
synthesis of anisotropic silver nanostructures, which may represent
promising tools as effective antimicrobial agents and for in vitro
diagnostics. The synthesized hollow nanostructures may be used for
enhanced drug encapsulation and sustained release.
[0175] Particular embodiments of the invention are described
below:
[0176] Methods that control of size and morphology of synthesized
nanostructures that do not require the use of particular polymers,
surfactants and/or special laboratory equipment. These methods are
used for synthesizing novel silver nanostructures having the
following characteristics:
[0177] sizes ranging from 0.17 to 7 .mu.M having distributed pores
ranging in size from 10 to 20 nm and that have a 3 dimensional
("3d") flower-like structure with multilayer hollows, rough
surfaces, external channels and distributed interior hollows;
[0178] unique flower like silver structures with multi layer of
hollows in the range of 3-15 layers, surface roughness ranged from
10-200 nm, more rough surface, with larger holes (10-20 nm in width
and 200-300 nm in length), highly external channels in the range of
1-4 channels surrounding silver structures with size ranging
between 0.2-3.0 .mu.M. Likewise 3d flower-like silver structures
with size in the range of 0.25-2.5 .mu.M with more multi layers
(16-30 layers), surface roughness ranged from 250-400 nm, with
larger holes ranged from 50-200 nm, highly external channels ranged
from 5-10 channels surrounding silver particles.
[0179] unique 3d shell-like silver structures with little pores
ranged between 5-30 nm, rough surface (180-300 nm), highly external
arms ranged from 3-9 arms surrounding particles, having size
ranging between 0.15-1 .mu.M.
[0180] unique scaffold like silver structures having size ranging
between 2-7 .mu.M with highly inter connected pores ranging between
50-100 nm width and 100-500 nm length;
[0181] unique roll fiber twin like silver structures having size
ranging between 0.15-1 .mu.M with multi hard external arms on the
surface complete shelled the particles;
[0182] unique 3d pores spherical silver structures have size in the
range 0.6-5 .mu.M of with well distributed external pores on the
surface ranges between 50 nm-300 nm;
[0183] unique 3d pores spherical silver structures spongy like with
size in the range of 0.3-2 .mu.M with well interconnected pores in
the range of 20-100 nm;
[0184] flower like with multi external interacted layers of
hollows, branched rough edges silver nanostructures with size in
the range of 0.3-1.5 .mu.M with well controlled size and
dispersion;
[0185] flower-like silver structures with multi layer of paper,
hollows like cores, soft surface silver nanostructures with size in
the range of 0.2-1 .mu.M with well controlled size and
dispersion;
[0186] trees with multi branched edges like arms like silver
nanostructures with size in the range of 2-6 .mu.M with well
controlled size and dispersion;
[0187] dendrimer silver nanostructures with size in the range of
0.7-2 .mu.M with well controlled size and dispersion;
[0188] flower silver structures with more internal hollows, rough
surface silver nanostructures with size in the range of 0.24-1.5
.mu.M with well controlled size and dispersion;
[0189] cubes silver structures with size in the range of 0.1-1
.mu.M with well controlled walls and edges;
[0190] pyramidal silver structures with size in the range of 0.05-2
.mu.M with well controlled walls, edges and soft surface;
[0191] bibode silver structures with size in the range of 50-200 nm
with soft surface;
[0192] triangular silver structures with size in the range of 0.1-1
.mu.M;
[0193] octahedral multi layer silver structures with size in the
range of 100-400 nm;
[0194] octahedral multi layer, with pores on the surface silver
structures with size in the range of 100-300 nm;
[0195] ribbed like silver structures with size in the range of
0.3-1 .mu.M;
[0196] multi ribbed with cubes decorated on the surface silver
structures with size in the range of 100-600 nm;
[0197] octahedral with cube decorated on the surface silver
structures with size in the range of 0.2-1 .mu.M;
[0198] silver stars with size in the range of 0.1-4 .mu.M and with
soft branched arms and hole as core like;
[0199] silver stars with size in the range of 0.15-3 .mu.M and with
soft and rough, branched, more layer of arms and hole as core
like;
[0200] silver flower structures with size in the range of 100-400
nm with soft, more layers of arms;
[0201] silver myriad dendrimer structures with size in the range of
0.5-2 .mu.M, with highly branched arms and core like;
[0202] silver butterfly structures with size in the range of
0.6-1.5 .mu.M and with highly branched arms like wings;
[0203] silver stars, flower structures with size in the range of
0.3-2 .mu.M and by incorporation of polymers with TSC and using UV
irradiation; and
[0204] silver stars, flower structures with size in the range of
0.2-1 .mu.M and by incorporation of polymers with TSC and without
using of UV irradiation.
Synthesis of Anisotropic Silver Structures
[0205] The anisotropic silver structures silver nanostructures, and
microstructures with control in size, shape were synthesized by
chemical reduction of silver nitrate with dextrose in presence of
trisodium citrate and sodium hydroxide (NaOH). Dextrose acts as
reducing agent, as capping agents TSC act only as capping material,
NaOH acted enhance reduction efficacy of dextrose and as shape
control.
[0206] Likewise, cubes, pyramidal, triangular, silver
nanostructures, macrostructures, are prepared by incorporation of
ascorbic acid with TSC, dextrose and NaOH. By the same way dextrose
act as reducing agent, as capping agents TSC act only as capping
material, NaOH acted enhance reduction efficacy of dextrose,
Ascorbic acid and as shape control. The particles morphology, size
were controlled by reaction condition include amount of TSC,
AgNO.sub.3, dextrose, ascorbic acid, and NaOH as will be
demonstrated and discussed below. The methods demonstrated herein
provide nanostructures, macrostructures with high uniformity in
size, controllable size, morphology, large quantities, reproducibly
and good solubility in various solvents. In addition to such method
are fast, echo-friendly, and inexpensive, with lack of specially
laboratory equipments and laboratory skills.
[0207] A round bottom glass flask (100 mL) were cleaned in aqua
regia (3 parts HCl, 1 part HNO.sub.3) and rinsed with DDI water and
dried in the oven at 60.degree. C. All the other glasses were
should be cleaned by the same way.
[0208] The formulas included silver nitrate AgNO.sub.3 (silver
source), trisodium citrate salts, anhydrous (TSC) (shape control
reagents), dextrose (reducing agents, shape controller), L-Ascorbic
acid (ascorbic acid) as reducing agents, Sodium hydroxide pellets,
97% (NaOH) and DDI water as solvents all above reagents were
provided from Sigma Aldrich and water provided from mille pore
(Millipore Corporation, Billerica, Mass.) with a resistivity of 18
M.OMEGA.cm in our lab.
[0209] A solution of AgNO.sub.3 were prepared by dissolved
appropriate amount of AgNO.sub.3 in DDI water with final
concentrations of 0.001-2.0 M and 0.001-0.1 mM, and vortex for 1.5
min to complete dissolution and covered with aluminum fuel to
protect from light. A solution of TSC was prepared by dissolving
appropriate amount of TSC in DDI water with final concentrations of
0.001-2.0M and vortex for 1.5 minute to complete dissolution. A
solution of Ascorbic Acid were prepared by dissolved appropriate
amount of Ascorbic Acid in DDI water with final concentrations of
0.3-3.0 M and vortex for 2 min to complete dissolution and covered
with aluminum fuel to protect from light. A solution of dextrose
were prepared by dissolved appropriate amount of dextrose in DDI
water with final concentrations of 0.001-2.0 M and vortex for 2 min
to complete dissolution and cover with aluminum fuel to protect
from light. A solution of NaOH was prepared by dissolved
appropriate amount of NaOH in DDI water with final concentrations
of 0.001-5.0 M and vortex for 2 min to complete dissolution. All
the above reagents should be stored in sterilized Falcon tubes.
Synthesis of Nanoparticles Having 3d Flower-Like Morphology
[0210] The 3d flower like morphology with distributed interior
hollow structures; branched edges are synthesized by the chemical
reduction of silver nitrate in aqua's phase. Briefly 0.001-2.0 M
AgNO.sub.3, 0.001-2.0M TSC solution, 0.3-3.0M of dextrose added to
15 mL DDI, then added various concentration of NaOH 0.001-5.0 M,
stirred at room temperatures the color change immediately to deep
gray or green, deep yellow depended on the reaction condition after
the color changed, the solution is stirred for an additional 5
minutes, and stirrer turn off. The particles were collected by
centrifugation at 1400 rpm for 10 minute, the supernatant was
discarded and precipitate re-suspended in DDI water; the process
was repeated three times to remove excess dextrose.
Example 1
[0211] The 3d flower-like silver structures with multi layer of
hollow, rough surface, external channels surrounded particles were
synthesized according to the above condition but using 0.2 mL of
AgNO.sub.3, 0.4 mL of TSC, 0.4 mL of dextrose added to 15 mL of DDI
water; stirring at room temperature the color turned to deep yellow
immediately after addition of 100 .mu.L of NaOH then the solution
is stirred for an additional 5 min, stirrer turned off and samples
collected by centrifugation as mentioned above (FIG. 8).
Example 2
[0212] The 3d flower-like silver structures with more multilayer of
hollow, more rough surface, with larger hollows, highly external
channels surrounded particles were synthesized according above
condition but using 0.2 mL of AgNO.sub.3, 0.4 mL of TSC, 0.4 mL of
dextrose added to 15 mL of DDI water, stirring at room temperatures
the color turn to gray immediately after addition of 250 .mu.L of
NaOH then the solution is stirred for an additional 5 minutes,
stirrer turned off and samples collected by centrifugation as
mentioned above (FIG. 9).
Example 3
[0213] The 3D scaffold fibers like, flakes, and cluster silver
structures little hollow pores, more rough surface, highly external
arms surrounded particles were synthesized according above
condition but using 1 mL of AgNO.sub.3, 1 mL of TSC, 1 mL of
dextrose added to 15 mL of DDI water, stirring at room temperatures
the color turn to green immediately after addition of 50 .mu.L of
NaOH then the solution is stirred for an additional 5 min, stirrer
turned off and samples collected by centrifugation 3 times at
8,000, 12,000, and 14,000 rpm, respectively.
Claim 80; Example 3 at page 16 the word (3D shell-like) should be
substituted by (3D scaffold fibers like, flakes, and cluster).
Also, the following statement should be added centrifugation 3
times at 8,000, 12,000, and 14,000 rpm respectively (calim80)
Example 4
[0214] The 3d scaffold-like silver structures with little pores
hollow, more rough surface, were synthesized according above
condition but using 0.3 mL of AgNO.sub.3, 0.6 mL of TSC, 0.6 mL of
dextrose added to 15 mL of DDI water, stirring at room temperatures
the color turn to deep green immediately after addition of 100
.mu.L of NaOH then the solution is stirred for an additional 5
minutes, stirrer turned off and samples collected by centrifugation
as mentioned above (FIG. 11).
Example 5
[0215] The 3d roll fiber twin-like silver structures with little
pores hollows, more rough surface, were synthesized according above
condition but using 0.3 mL of AgNO.sub.3, 0.6 mL of TSC, 0.6 mL of
dextrose added to 15 mL of DDI water, stirring at room temperatures
the color turn to deep green immediately after addition of 250
.mu.L of NaOH then the solution is stirred for an additional 5 min,
stirrer turned off and samples collected by centrifugation as
mentioned above (FIG. 12).
Example 6
[0216] The porous spheroid silver structures with little pores
hollow, more rough surface, were synthesized according above
condition but using 1 mL of AgNO.sub.3, 1 mL of TSC, 1 mL of
dextrose added to 15 mL of DDI water, stirring at room temperatures
the color turn to deep green immediately after addition of 200
.mu.L of NaOH then the solution is stirred for an additional 5
minutes, stirrer turned off and samples collected by centrifugation
as mentioned above (FIG. 13).
Example 7
[0217] The porous spherical sponge-like silver structures with high
inter connected pores, more rough surface, were synthesized
according above condition but using 1 mL of AgNO.sub.3, 1 mL of
TSC, 1 mL of dextrose added to 15 mL of DDI water, stirring at room
temperatures the color turn to deep green immediately after
addition of 100 .mu.L of NaOH then the solution is stirred for an
additional 5 minutes, stirrer turned off and samples collected by
centrifugation as mentioned above (FIG. 14).
Example 8
[0218] The 3d flower-like silver structures with multi external
interacted layers of hollow, branched rough edges were synthesized
according to above condition but using 1 mL of AgNO.sub.3, 1 mL of
TSC, 1 mL of dextrose added to 1 omL of DDI water, added 50 .mu.L
of NaOH stirring with heating up to 100.degree. C. the color turn
to yellow to deep brown immediately after 5 min then the solution
is stirred till to cooling to room temperatures and stirrer turned
off and samples collected by centrifugation as mentioned above
(FIG. 15).
Example 9
[0219] The flower like silver structures with multi layer of paper,
hollow-like cores, soft surface, were synthesized according to
above condition but using 2.0 mL of AgNO.sub.3, 2 mL of dextrose,
stirring at room temperatures the color turn to deep yellow
immediately after addition of 20 .mu.L of NaOH then the solution is
stirred for additional 10 min samples collected by centrifugation
as mentioned above (FIG. 16).
Example 10
[0220] The treelike silver structures with multi branched edges
like arms were synthesized according to above condition but using 1
mL of AgNO.sub.3, 2 mL of dextrose, added to 10 mL DDI water, then
added 100 .mu.L of NaOH stirring with heating up to 100.degree. C.
the color turned to yellow to deep gray with precipitates after 3
min then the solution was allowed to cool to room temperature with
stirring and samples collected by centrifugation as mentioned above
(FIG. 17).
Example 11
[0221] The flower-like silver structures with more wide internal
hollow like core, multi-edge like arms were synthesized according
to above condition but using 2 mL of AgNO.sub.3, 1.0 mL of TSC, 1.0
mL of dextrose, then added 50 .mu.L of NaOH and stirring with
heating up to 100.degree. C. the color turned to yellow to deep
yellow after 5 min then the solution was stirred till cooling to
room temperature and samples collected by centrifugation as
mentioned above (FIG. 18).
Example 12
[0222] The dendrimer silver structures were synthesized according
to above conditions but using 0.5 mL of AgNO.sub.3, 0.5 mL of
dextrose, added to 5.0 mL of DDI water, then added 100 .mu.L of
NaOH with stirring at room temperature the color turned to yellow
to deep gray and a precipitate was formed immediately, then the
solution was stirred for additional 5 min and samples collected by
centrifugation as mentioned above (FIG. 19).
Example 13
[0223] The unique flower silver structures with more internal
hollow, rough surface were synthesized according to above
conditions but using 1.0 mL of AgNO.sub.3, 1.0 mL of TSC, 1.0 mL of
dextrose, then added 50 .mu.L of NaOH, then stirring with heating
up to 100.degree. C., the color turned to yellow to deep gray then
the solution was stirred till cooling to room temperature, and
samples collected by centrifugation as mentioned above (FIG.
20).
Example 14
[0224] The cube silver structures were synthesized according to
above condition but using 0.4 mL of AgNO.sub.3, 0.4 mL of dextrose,
0.4 mL of TSC, added to 5 mL of DDI water, then added 200 .mu.L of
ascorbic acid, then added 200 .mu.L of NaOH and stirring at room
temperature the color turned to yellow to deep gray immediately,
then the solution was stirred for additional 5 min and samples
collected by centrifugation as mentioned above (FIG. 21).
Example 15
[0225] The pyramidal silver structures were synthesized according
to above condition but using 0.5 mL of AgNO.sub.3, 0.5 mL of
dextrose, added to 5.0 mL of DDI water, then added 300 .mu.L of
NaOH stirring at room temperature the color turned to yellow to
deep gray and a precipitate formed immediately, then the solution
was stirred for additional 5 min and samples collected by
centrifugation as mentioned above (FIG. 22).
Example 16
[0226] The bibode silver structures were synthesized according to
above conditions but using 1.0 mL of AgNO.sub.3, 1.0 mL of
dextrose, then added 150 .mu.L of NaOH and stirring at room
temperature the color turned to deep gray immediately, then the
solution was stirred for additional 5 min and samples collected by
centrifugation as mentioned above (FIG. 23).
Example 17
[0227] The triangular silver structures were synthesized according
to above conditions but using 1.0 mL of AgNO.sub.3, 1.0 mL of TSC,
1.0 mL of dextrose added 100 .mu.L of NaOH stirring with heating up
to 100.degree. C. the color turned to yellow to deep gray
immediately after 5 min then the solution was stirred till cooling
to room temperature and samples collected by centrifugation as
mentioned above (FIG. 24).
Example 18
[0228] The octahedral multilayer silver structures were synthesized
according to above condition but using 0.5 mL of AgNO.sub.3, 0.75
mL of dextrose, then adding 300 .mu.L of ascorbic acid, stirring at
room temperature the color turn to deep gray immediately, then the
solution was stirred for additional 5 min and samples collected by
centrifugation as mentioned above (FIG. 25).
Example 19
[0229] The octahedral multi layer, with pores on the surface silver
structures were synthesized according to above condition but using
0.5 mL of AgNO.sub.3, 0.75 mL of dextrose, then added 200 .mu.L of
ascorbic acid, stirring at room temperatures the color turned to
deep gray immediately, then the solution was stirred for an
additional 5 min and samples collected by centrifugation as
mentioned above (FIG. 26).
Example 20
[0230] The ribbed silver structures were synthesized according to
above conditions but using 0.5 mL of AgNO.sub.3, 0.75 mL of
dextrose, then added 150 .mu.L of ascorbic acid, stirring at room
temperatures the color turn to deep gray immediately, then the
solution was stirred for additional 5 min and samples collected by
centrifugation as mentioned above (FIG. 27).
Example 21
[0231] The synthesis of multi ribbed with cubes decorated on the
surface silver structures was done according to above conditions
but using 0.5 mL of AgNO.sub.3, 0.75 mL of dextrose, then adding
100 .mu.L of ascorbic acid, stirring at room temperatures the color
turned to deep gray immediately, then the solution was stirred for
an additional 5 min and stirrer turn off and samples collected by
centrifugation as mentioned above (FIG. 28).
Example 22
[0232] The synthesis of octahedral with cube decorated on the
surface silver structures was carried out according to above
conditions but using 0.5 mL of AgNO.sub.3, 0.75 mL of dextrose,
added to 10 mL DDI water, then adding 100 .mu.L of ascorbic acid
and 100 .mu.L of NaOH directly after ascorbic acid and stirring at
room temperature. The color turned to pale gray immediately, then
the solution was stirred for an additional 5 min and samples
collected by centrifugation as mentioned above (FIG. 29).
Synthesis of Silver Stars, Flower, Myriad Dendrimer and
Butterfly
[0233] Stars, flower, myriad dendrimer, butterfly silver structures
fly have been synthesized by reduction of AgNO.sub.3 in aqua's TSC
solution, TSC with polymer solution by ascorbic acid at room
temperature. Likewise reduction of AgNO.sub.3 in aqueous TSC with
polymers was achieved by UV and then by ascorbic acid. The particle
morphology and size were manipulated by changing amounts of
AgNO.sub.3, TSC, and duration of UV irradiation.
Example 23
[0234] The silver stars with soft arms were synthesized by the
chemical reduction of silver nitrate in aqua's phase. Briefly; 0.3
mL of 0.001-0.1 mM AgNO.sub.3, 0.5 mL of a 0.0001-2.0M TSC solution
were added to 10 mL DDI, followed by 100 .mu.L of ascorbic acid
(0.3-3.0 M) and stirred at room temperature. The color changed
immediately to gray and the solution was stirred for an additional
5 min, and stirrer turn off. The varying amounts of TSC (0, 300,
400, 500, 600, 700, 800, 1100 .mu.L) lead to formation of silver
stars with soft arms as shown in FIG. 30 A-H.
Example 24
[0235] Silver stars with soft and rough, branched, more layer of
arms and holes as core like were formed by the same conditions
above but using (50, 100, 200, 300, 400, 500, 600, 700, 800 .mu.L)
of 0.001-0.1 mM AgNO.sub.3 and using 0.5 mL of a 0.0001-2.0M TSC
solution added to 10 mL DDI, then adding 100 .mu.L of ascorbic acid
0.3-3.0M, and stirred at room temperature. The color change
immediately to gray. After color change, the solution was stirred
for an additional 5 min. The color changed faster by using higher
amounts of AgNO.sub.3 FIG. 31 A-I.
Example 25
[0236] Silver flower structures were formed by the same condition
above but using 50, 100, 150, 200, 250, or 300 .mu.L of 0.3-3.0 M
ascorbic acid added to 10 mL DDI containing 300 .mu.L 0.001-0.1 mM
AgNO.sub.3 and using 0.5 mL of 0.0001-2.0M TSC solution, then
stirred at room temperature. The color changed immediately to gray,
the solution is stirred for an additional 5 min. The color changed
faster by using higher amounts of ascorbic acid FIG. 32 A-F.
Example 26
[0237] Silver myriad dendrimer structures were synthesized by the
same conditions above and varying the amounts of TSC and
AgNO.sub.3. Briefly 300, 200, or 100 .mu.L of 0.0001-2.0 M TSC were
added to 300, 200, or 100 .mu.L of 0.001-0.1 mM AgNO.sub.3; and 10
mL DDI then 100 .mu.L of 0.3-3.0M ascorbic acid were added. After
stirring at room temperature the color changed to gray then the
solution was stirred for an additional 5 min. The color changed
faster when higher amounts of TSC and AgNO.sub.3 were used FIG. 33
A-C.
Example 27
[0238] Silver butterfly were synthesized by the same condition
above but using 500 .mu.L of 0.0001-2.0 M TSC and 300, 200, or 100
.mu.L of 0.001-0.1 mM AgNO.sub.3 with 10 mL DDI then adding 80, 100
and/or 150 .mu.L of 0.3-3.0 M ascorbic acid. After stirring at room
temperature the color changed to gray then the solution was stirred
for an additional 5 min. The color changed faster when higher
amounts of ascorbic acid and AgNO.sub.3 were used FIG. 34 A-C.
Example 28
[0239] Silver stars were synthesized by using 0.001-0.1 mM
AgNO.sub.3, 0.5 mL of a 0.0001-2.0M TSC, 0.3 mL of polymer solution
(PEG, PVA, PEI,) added to 10 mL DDI, then subjected to 18 W
ultraviolet light at wave length (430 nm) for 30 min. The color
turned to gray gradually. After adding 100 .mu.L of 0.3-3.0 M
ascorbic acid at room temperature the gray color changed to deep
gray immediately. The solution was stirred for an additional 5 min.
PVA or PMMA have been used with TSC as morphology controlling
gannet for synthesis of star like silver structures with branched
arms. Briefly, 0.3 ml of polymer solution (PVA or PMMA) with mixed
with AgNO.sub.3, TSC and reduced with ascorbic acid with and
without UV (claim 15). The varied time of UV irradiation have noted
effects on the size and morphology of the synthesized particles
FIG. 35 A-C.
Example 29
[0240] Silver stars, were synthesized without UV irradiation by
using 0.3 mL of a 0.001-0.1 mM AgNO.sub.3, 0.5 mL of a 0.0001-2.0 M
TSC, 0.3 mL of polymer solution such as (PEG, PVA, PEI) added to 10
mL DDI. 100 .mu.L of 0.3-3.0M ascorbic acid was added and stirred
at room temperature the gray color change gradually to deep gray
immediately, after the color changed, the solution is stirred for
an additional 5 min. PVA or PMMA have been used with TSC as
morphology controlling gannet for synthesis of star like silver
structures with branched arms. Briefly, 0.3 ml of polymer solution
(PVA or PMMA) with mixed with AgNO.sub.3, TSC and reduced with
ascorbic acid with and without UV (claim 15). The varied amount of
TSC, AgNO.sub.3, polymers have noted effects on the size and
morphology of the synthesized particles as shown in FIG. 36
A-C.
[0241] Zeta potential for silver structures (Example 1-7 and 23-29)
were measured with dynamic light scattering NANO ZS Malvern zeta
sizer equipment (Worcestershire, UK), at 25.degree. C., using a
He--Ne laser of 633 nm wavelength and a detector angle of
173.degree.. Four independent measurements were made for each
sample as tabulated in table 1. The result clearly demonstrated
that, the silver structures are coated with negative charge ranged
between -33 and -65 which it is strong enough to protect it from
aggregation. Therefore the presence of electrical charge on the
surface of silver structures makes it a promising tool in clinical
diagnostics.
Example 30
[0242] See attached "Controlled synthesis and characterization of
hollow flower-like silver nanostructures".
[0243] Only the polyol process was versatile for the synthesis of
the following structures: spherical, cubes, pyramidal, hollow
cubes, bars, rice-like, octahedral, beam and spheroid. Polyol
method gives mixtures of structures and there is a need for
separation of different structures by nanofiltration.
[0244] Methods using poly (lactic-co-glycolic) acid templates gave
similar star-like structures to the method of the invention but
were not observed to provide the same variety of different
structures. The method also requires a polymer template with or
without UV irradiation and generates large size of stars, with lack
of size control.
[0245] The nanoparticles of the invention can be used in numerous
different applications.
[0246] They may be further processed, for example, by application
of a surface coating to modify their solubility in polar or
nonpolar media. For many biological applications, the application
of a polar surface coating would be advantageous to provide
solubility in aqueous media or in biological fluids. Nanoparticles
may be functionalized with particular molecules to target them to
particular receptors or to track their distribution. Examples of
such targeting moieties include antibodies, protein or nucleic acid
ligands that bind to specific receptors or molecules, aptamers, or
other tags such as radioactive agents, fluorescent dyes, or tags
like (strept)avidin or biotin or nickel and histidine. Hollow
nanoparticles or nanoparticles having hollow portions can be used
as vessels for carrying or containing other molecules such as those
useful for imaging, plasmonics, or biosensing.
[0247] Silver spherical nanoparticles (FIG. 68) have been
encapsulated inside liposome nanoparticles to act as a sustained
broad spectrum antibacterial agent. The kinetic release,
cytotoxicity, and antibacterial against Gram negative and Gram
positive Bactria namely; Escherichia coli, Salmonella enterica,
Pseudomonas aeruginosa, and Staphylococcus aureus were investigated
against as described in the paper [Sustained Broad Spectrum
Antibacterial Effects of Nanoliposomes Loaded with Silver
Nanoparticles]. The strains growth was inhibited by more than 80%
upon usage of using 200-225 .mu.M of silver nanoparticles. The
results obviously revealed that, the silver nanoparticles are
promising tools in antibacterial therapy and wound healing. The
optical properties of the silver nanoparticles were investigated by
UV-visible-NIR measurements (Perkin Elmer UV/Vis/NIR Win lab lambda
950, 950N6102502, UK) and Pro Raman-L Analyser (Enwave Optronics
Inc. 18200 McDurmott Calif. 92614, USA). FIGS. 33-61 show the
UV/Vis/NIR extinction spectra of the silver structures. The
spectrum of the silver structures exhibit all characteristic peaks
corresponding to different modes of plasmon excitation of all
structures. There is more than dominant one band, located at around
342-300 nm and with intensity ranged between 500-13,000 a.u.
Therefore the ability of silver structures to located band in both
UV and NIR makes it a promising tool in SERS, optical, electronic,
sensors and plasmonic applications.
Example 31
[0248] Dextrose was used to reduce silver nitrate with heating to
form spherical silver nanostructures FIG. 37. Briefly, the
spherical morphology was synthesized by the chemical reduction of
silver nitrate in aqua's phase. An amount of 0.001-2M AgNO.sub.3
solution mixed with 0.1-2 mM of dextrose and stirred with heating
the color change immediately to deep yellow. After the color
changed, the solution is stirred for an additional 5 minutes, and
stirrer turn off. The particles collected by centrifuge at 1400 rpm
for 10 minute, the supernatant can be removed, precipitate are
re-suspended in DDI water and repeat three time for remove excess
of dextrose. The Silver particle having size in the range of 10-50
nm and particle size depended on the concentration of dextrose.
TABLE-US-00001 TABLE 1 Zeta potential for all silver structures
samples 1-29. Zeta potential Examples mV 1 -45 .+-. 2 2 -35 .+-. 3
3 -28 +3 4 -55 .+-. 4 5 -37 .+-. 2 6 -44 .+-. 5 7 -33 .+-. 4 23 -65
.+-. 3 24 -55 .+-. 2 25 -48 .+-. 4 26 -62 .+-. 5 27 -59 +4 28 -61
.+-. 3 29 -48 .+-. 3
TABLE-US-00002 TABLE 2 Other Versatile Methods for Synthesis of
Silver Nanoparticles: Morphology Methods Reagents of Structures
Reference Citrate Materials: Spherical, quasi [1-7] reduction
AgNO.sub.3 sphere and, 2-propanol octahedral., wire N.sub.2O and
triangle Sodium citrate (stabilizer) pH controller such as NaOH UV
Polyol Materials Spherical, wires, [8-25] (ethylene AgNO.sub.3 or
(CF.sub.3COOAg) beam, rice, cubes, [26] glycol + Ethylene glycol
(solvent- bars, bipyramid, PVP) reducing agent) beam, octahedron,
PVP (stabilizer polymer). cube, truncated Reducing agent: NaBr
octahedron, and HNO.sub.3 tubes NaHs with HCl CuCl, FeCl.sub.3
formamide/ethanol Light- Material: Silver chains, [7, 27-33]
Mediated AgNO.sub.3 polygonal plates, Synthesis Methoxy
polyethylene glycol disk, wire, rods or photosensitive polymer as
and octahedron, Template Ethanol solution spherical Hyperbranched
polyurethane UV Polyol reagent Seed Materials: rods, wire, [6, 30,
34- growth AgNO.sub.3 branched, 40] Sodium citrate, decahedron and
L-arginine, cubes PVP NaBH4 Ascorbic acid PtCl or other metal salt
CTAB or BDAC or combination of two
Example 32
AgNPs Encapsulated in Nanolipososmes as Effective Broad Spectrum
Anti-Microbial Agents
[0249] Nanoliposomes (<50 nm) were prepared using a modified
reverse phase evaporation method and spherical dextrose-capped
AgNPs were synthesized. The prepared liposome AgNPs (LAgNPs) were
characterized and tested for their antibacterial effects. The size
of LAgNPs is 25-80 nm. Release of AgNPs from nanoliposomes was
sustained over 10 h. Complete growth inhibition of Eschericia coli,
Salmonella enterica, Pseudomonas aeruginosa, and Staphylococcus
aureus was achieved using 180, 200, 160, and 120 .mu.M,
respectively, of LAgNPs. As shown below, LAgNPs exhibited sustained
broad-spectrum antibacterial effects as compared to free AgNPs.
[0250] Synthesis of spherical silver nanoparticles. Silver nitrate
(AgNO.sub.3), dextrose, egg Phosphatidylcholine (PC), cholesterol
(Cho), MacConckey agar medium, broth medium, agarized Czapek Dox,
ethanol, chloroform, and sodium hydroxide (NaOH) were purchased
from Sigma-Aldrich Chemie GmbH (Munich, Germany). HCl and HNO.sub.3
were purchased from El-Gomhouria Co, (Cairo, Egypt). Double
deionized water (DDI) was prepared using a Milli-Q.TM. system
(Direct-Q 3, Model ZRQSOPOWW, Millipore Corporation, Billerica,
Mass.) with a resistivity of 18 M.OMEGA.cm.
[0251] AgNPs were synthesized by the chemical reduction of
AgNO.sub.3 in aqueous solution. Briefly, a round-bottom flask was
cleaned thoroughly with aqua regia (3HCl: 1HNO.sub.3) then rinsed
with DDI water. AgNO.sub.3 (0.01-1.0 M) was added to 0.1-2.0 M
dextrose solution and dissolved in water at room temperature. NaOH
(0.001-0.2 mM) was added during stirring. The solution changed from
colorless to yellow, brown or green depending on the amount of
AgNO.sub.3, dextrose, and NaOH. Following color change, the
solution was stirred for an additional 5 min, centrifuged, and
washed three times with DDI water to remove excess dextrose.
[0252] Nanoliposomes were prepared using PC and Cho with different
molar ratios (1:1, 1:2, 1:3, 1:4, 1:5, and 1:6). The lipid
components were dissolved in 5 mL of chloroform and ethanol mixture
(6:1, v/v). The solvents were removed using a rotary evaporator
(Buchi RE-111 Rotavapor, Brinkmann, Westbury, N.Y.) at 55.degree.
C., 25 rpm, and high vacuum which has resulted in a lipid thin
film. The film was redissolved in 10 mL PBS, pH 7.4 and the mixture
was vortexed for 2 min and then sonicated using a probe sonicator
(Model GM 2200, Bandelin Electronic, Berlin, Germany) with heating
to form vesicles. The undispersed vesicles (aggregates) were
separated by filtration.
[0253] For the synthesis of LAgNPs, the thin films prepared above
were rehydrated with 10 mL PBS, pH 7.4, containing AgNPs (5 nm).
The mixture was vortexed and sonicated with heating and then
incubated at room temperature for 3 h then filtered to remove
aggregates and free unencapsulated AgNPs
[0254] Mean particle size diameter and polydispersity index of
nanoliposomes and LAgNPs were measured directly after synthesis,
using photo correlation spectroscopy (Malvern Instruments Ltd,
Worcestershire, UK).
[0255] The size and morphology of the synthesized nanostructures
were studied using scanning electron microscope (SEM, LEO SUPRA 55;
Carl Zeiss AG, Oberkochen, Germany) and transmission electron
microscope (TEM, JEOL X100, Japan). Briefly, silver samples were
mounted on silicon wafer coated with aluminum foil and left 2 hours
to dry before imaging without sputter coating before SEM imaging at
an accelerating voltage of 6 kV and magnification of 150-200 k X.
Liposome samples were diluted with BPS and sonicated for 3 min then
negatively stained with 2% uranyl acetate and mounted on TEM grids
(carbon film supported by a copper grid) and allowed to dry for 2 h
before imaging with TEM at an accelerating voltage ranging from
200-220 kV and magnification of 400-500 kX. The particle size was
reported as the mean diameter of randomly selected structures.
[0256] The encapsulation efficiency of AgNPs in liposome was
measured using atomic absorption spectrophotometer (Z-5000,
Hitachi, Ltd., Tokyo, Japan). Briefly, 5 mL of the synthesized
LAgNPs were injected into the system and the percentage of
encapsulated AgNPs was calculated as follows: Silver
loading=[(WT-WS)/WT].times.100%, where WT is the total AgNPs added
to the liposomes and WS is the portion of AgNPs that was not
encapsulated and present in the supernatant after
ultracentrifugation of LAgNPs.
[0257] Different preparations of LAgNPs (PC:Cho; 1:1, 1:2, 1:3)
loaded with AgNPs (5 nm) were placed in cellulose dialysis bags.
The bags were suspended in 30 mL of PBS, pH 7.4 where AgNPs were
released into the buffer by diffusion. The release of AgNPs from
the dialysis bags was observed over a period of 10 h. Each hour, 1
mL of the buffer was removed, and substituted with fresh buffer, to
measure the concentration of released AgNPs using atomic absorption
spectrophotometer equipped with silver lamp (Z-5000, Hitachi, Ltd.,
Tokyo, Japan). The instrument parameters were: 328.1 nm wavelength;
5 mA lamp current; 0.5 nm band pass, fuel flow rate 0.9-1.2 L/min,
and temperature of 1100.degree. C.
[0258] Escherichia coli, Salmonella, P. aeruginosa, and S. aureus
were obtained from the department of Microbiology, VACSERA, Cairo,
Egypt. Bacterial cells were cultured for 24 h on a MacConckey agar
plates at 37.degree. C. Colonies were resuspended in LB broth
medium to achieve 10.sup.6 CFU/mL. This has been confirmed by
measuring bacterial growth as optical density using a microplate
plate reader (Tecn Infinity M200, CA, USA). Different
concentrations of free AgNPs, LNPs (1PC:3Cho), or LAgNPs
(1PC:3Cho:1AgNP) were added to the bacterial cultures. The
bacterial growth in the culture medium was monitored by measuring
the optical density at 700 nm.
[0259] The optical density of bacterial cultures, grown in 3
replicates with shaking at 37.degree. C. with and without
nanoparticles, was recorded every 60 min. The rate of bacterial
growth was calculated using the following formula:
[(Nt-No)/No.times.100]; where N.sub.o was the OD of bacteria at
time zero and N.sub.t is the OD of bacteria at the indicated time
point.
[0260] Table A illustrates particle size and polydispersity of
nanoliposomes and LAgNPs using photon correlation spectroscopy.
TABLE-US-00003 LNPs Size Poly LAgNPs PC:Cho (nm) Dispersity Size
(nm) Poly Dispersity 1:1 240 .+-. 35 0.13 25 .+-. 4 0.01 1:2 270
.+-. 25 0.16 45 .+-. 5 0.015 1:3 310 .+-. 22 0.18 50 .+-. 3 0.02
1:4 340 .+-. 38 0.2 65 .+-. 1 0.04 1:5 410 .+-. 40 0.31 70 .+-. 3
0.05 1:6 450 .+-. 28 0.4 80 .+-. 5 0.07 *Same molar ratio of AgNPs
was added to all PC/Cho preparations.
[0261] The LAgNPs size ranged between 25 to 80 nm and the size of
nanoliposomes between 250 to 400 nm. The SD values for LAgNPs were
calculated from four independent measurements. AgNPs of 5.+-.1 nm
were used. Nanoparticles showed low polydispersity indices (Table
A). The dynamic light scattering showed a narrow size distribution
of LAgNPs and poly size distribution for nanoliposomes. The
encapsulation of AgNPs into nanoliposomes has led to long-term
stability of LAgNPs.
[0262] The zeta-potential measurements of LAgNPs in solution ranged
between -76 and -58 mV. After 3 months of storage at room
temperature, measurements ranged between -69 and -45 mV. The
persistence of the negative charge on the LAgNPs is indicative of
their stability. The individual measurement results are shown in
the supplementary data file.
[0263] The size and shape of the synthesized AgNPs were studied
using SEM and particle size was reported as mean diameter of
randomly selected structures. FIG. 63A presents spherical silver
nanostructures with size in the range of 5.+-.1 nm. FIG. 63B
presents UV spectroscopy of AgNPs with maximum absorbance at 410
nm. FIG. 64 presents SEM (A-C) of nanoliposomes that were prepared
by using 1:1, 1:2, and 1:3 molar ratio of PC:Cho. The particles
have spherical morphology with particle size in the range of
250-400 nm.
[0264] The LAgNPs were analyzed by TEM (FIG. 64 D-E) and had
spheroid shape with an average particle size between 25-90 nm. The
addition of AgNPs to nanoliposomes may have contributed to the
reduced size of nanoliposomes to less than 50 nm. The presence of
charged AgNPs on the surface of nanoliposomes, supported by zeta
potential measurements of AgNPs (-38 mV) and LAgNPs (-76 mV)
(supplementary data), contributed to the stability of LAgNPs. This
result is in agreement with previous reports which demonstrated
that the presence of charged particles on the surface of
nanoliposomes contribute to their stability [21-22]. Other groups
used polymers or surfactants for the stabilization of liposomes
[21-23].
[0265] The amounts of AgNPs encapsulated into six different
formulas of nanoliposomes 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6 (PC:Cho)
were 60%, 75%, 88%, 80%, 78%, and 70%, respectively. The highest
encapsulation (88%) was achieved using nanoliposomes of
1PC:3Cho.
[0266] FIG. 65 shows the release kinetic of free AgNPs from LAgNP
prepared using 1:1, 1:2, and 1:3 PC:Cho. The rate of release of
AgNPs obeyed zero order kinetics with r.sup.2>0.96. This is
similar to the release patterns of other drugs from liposomes.
About 80% of the free AgNPs diffused out of the cellulose bag after
10 h. Release of AgNPs was 76%, 64%, 58% from liposomes prepared
using 1:1, 1:2, 1:3 PC:Cho, respectively. Therefore, sustained
release of AgNPs was observed using LAgNPs (1PC:3Cho; 50 nm).
Because of their high encapsulation efficiency and sustained
release of AgNPs, LAgNPs prepared using 1PC:3Cho were tested for
their anti-bacterial effects.
[0267] The antibacterial activity of LAgNPs prepared using 1PC:3Cho
was assayed against four common bacterial species namely: E. coli,
Salmonella, P. aeruginosa, and S. aureus and monitored by optical
density measurements using a micro-plate reader. Different
concentrations of the LAgNPs (20-225 .mu.M) were tested. LAgNPs
completely inhibited bacterial growth of E. coli (180 .mu.M),
Salmonella (200 .mu.M), P. aeruginosa (160 .mu.M), and S. aureus
(120 .mu.M) (FIG. 66A). The growth of the above strains was not
completely inhibited using 200-225 .mu.M of AgNPs (FIG. 66B).
Adding free nanoliposomes up to 150 .mu.M had no effect on
bacterial growth then the growth decreased by 30-45% upon
increasing the concentration of nanoliposomes to 225 .mu.M (FIG.
66C).
[0268] LAgNPs were more effective in inhibiting growth of Gram
positive bacteria (S. aureus) as compared to Gram negative bacteria
(E. coli, S. enterica, and P. aeruginosa). This may be due to the
difference in the structure of the outer walls of Gram positive and
negative bacteria.
[0269] Cytotoxicity of LAgNPs (0.05-0.3 mg/mL) to cultured human
fibroblast cells was investigated using MTT assay. LAgNPs
concentrations between 0.05 to 0.10 mg/mL were safe and did not
affect cell viability; higher LAgNPs concentrations (0.2-0.3 mg/mL)
were toxic to cultured fibroblasts.
[0270] This example shows for the first time that nanoliposomes
loaded with AgNPs are useful as broad-spectrum anti-bacterial
agents. The modified reverse phase evaporation method allowed the
preparation of nanoliposomes with size between 25-80 nm without the
need for high pressure homogenizer and extruder. Also, the AgNPs
were prepared by a green method and all reagents used for
preparation of nanostructures were non-toxic. The presence of AgNPs
stabilized the nanoliposomes and led to narrow size distribution of
LAgNPs. LAgNPs prepared with 1PC:3Cho demonstrated high
encapsulation capacity and provided long term release of AgNPs. The
results show the feasbility of using LAgNPs as a new generation of
antibacterial agents for sustained killing of multiple kinds of
bacteria. The LAgNPs are stable in pharmaceutical preparations and
provide long term anti-bacterial effect at the infection site.
* * * * *
References