Note: Descriptions are shown in the official language in which they were submitted.
Title DEVICE, METHOD, SYSTEM AND KIT FOR THE
DETECTION OF CONTAMINANTS AND/OR
PATHOGENS IN CONSUMABLES BY WAY OF A
COLOR-CHANGE ANALYSIS USING
NANOPARTICLES WITHIN A HYDROGEL
Inventors Attila Daniel TOTH
Maya TSELIOS
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FIELD OF INVENTION
[0001] The present invention relates to methods of detecting contaminants in
consumable
products such as foods and beverages.
BACKGROUND OF THE INVENTION
[0002] There is a significant public safety concern regarding food containing
adulterants,
pathogens, toxins, allergens and other impurities.
[0003] For example, there is concern regarding the adulteration of human food
and animal feed
stock by addition of triazine moieties, including melamine and cyanuric acid.
In 2008, melamine
and cyanuric acid contaminated infant formula resulted in the death and
hospitalization of
numerous infants in Asia. In China, six babies likely died and nearly 300,000
suffered urinary
problems from drinking melamine-tainted milk powder. In the United States in
2008, the Food
and Drug Administration (FDA) advised against human consumption of a number of
food
products including certain milk, chocolate, biscuit and coffee products
because of possible
melamine contamination. Furthermore, in 2008, the FDA reported finding trace
amounts of
melamine and cyanuric acid in infant formula marketed in the U.S.
[0004] In 2007, animal feed contaminated with melamine was discovered in fish
and livestock
fccd in the U.S. Additionally in 2007, pet food adulterated with melamine led
to the death of
roughly 5,000 animals in the United States and many more in other countries
outside the US,
including Europe and Canada.
[0005] In a 2009 study of 683 children diagnosed in Beijing in 2008 with
nephrolithiasis and
6,498 children without nephrolithiasis aged < 3 years, investigators found
that in children
exposed to melamine levels <0.2 mg/kg per clay, the risk for nephrolithiasis
was 1.7 times higher
than in those without melamine exposure, suggesting that the risk of melamine-
induced
nephrolithiasis in young children starts at a lower intake level than the
levels recommended by
the World Health Organization.
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[0006] As of July 2010, Chinese authorities were still reporting some seizures
of melamine-
contaminated dairy product in some provinces, though it was unclear whether
these new
contaminations constituted wholly new adulterations or were the result of
illegal reuse of
material from the 2008 adulterations.
[0007] In addition to melamine and cyanuric acid, significant public safety
risks are associated
with many other adulterants, pathogens, toxins and allergens contained in, for
example, human
food and animal feed.
[0008] Accordingly, there is an urgent need for devices and methods permitting
an untrained
user to rapidly and quantitatively detect harmful analytes that may be
contained in a variety of
different foods, beverages and other consumable products. It is an object of
the present
invention to obviate or mitigate the disadvantages of currently available
devices and methods,
which simply do not adequately address this consumer-self test market.
SUMMARY OF THE INVENTION
[0009] The present invention provides, in one aspect, a visually readable
sensor system for
detecting at least one of a presence of, absence of, or concentration of a
molecule of interest in a
consumable food and beverage products which system comprises: a cartridge for
collecting a
sample of the consumable product; a vessel, a hydrogel comprising an analyte
detecting
nanoparticle contained in the vessel, and a removable cover for covering the
hydrogel and the
vessel, wherein the nanoparticle is at least one of the following: directly
interactable and
indirectly interactable with the molecule of interest such that upon an
interaction, a change to
the physical configuration of the nanoparticles is created, which change in
configuration is a
visible to the naked eye.
[0010] The present invention provides, in another aspect, a method of
detecting at least one of a
presence of, absence of, or concentration of a molecule of interest in a
consumable sample, said
method comprising the steps of:
a. providing a pre-assembled, portable home test kit comprising a
substrate, a
hydrogel comprising an analyte detecting nanoparticle supported on the
substrate,
and a removable cover covering said hydrogel on said substrate,
b. removing the cover;
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c. exposing the consumable sample to a surface of a hydrogel; and
d. detecting interaction of the molecule of interest with the analyte
detecting
nanoparticle, such interaction producing change to a configuration of the
nanoparticles which is a visible to the naked eye.
[00111 The present invention provides, in another aspect, a pre-assembled,
portable home test kit
for the detection of molecule of interest in consumable food and beverage
products, which is
usable by a consumer in the absence of further analytical or detection
equipment and without
laboratory or analytical training, said kit comprising: a substrate, a
hydrogel comprising an
analyte detecting nanoparticle supported on the substrate, and a removable
cover covering said
hydrogel on said substrate, wherein said nanoparticle is at least one of the
following: directly
interactable and indirectly interactable with the molecule of interest such
that upon an
interaction, there is produced a change to a configuration of the
nanoparticles within the
hydrogel, said change in configuration being visible to the naked eye.
[0012] The present invention provides, in another aspect, a pre-assembled
comprehensive home
test kit to be used for testing raw or finished food or beverage products that
may be contaminated
with a molecule of interest. The home test kit, including simple, safe and
disposable materials, is
designed to be affordable, non-hazardous, straightforward and easy to use.
[0013] It would be particularly useful to be able to employ a simple,
convenient, discreet and
portable device, method and system to detect the presence or absence of
different substances in
foods and solutions such as beverages since a consumer may not be aware of
what he or she is
actually ingesting. The portable device, method and system of this invention
advantageously
enables the user to rapidly be able to determine whether a consumable being
purchased, served
or desired to be consumed comprises (or does not comprises) certain
substances, impurities or
adulterations therein.
[0014] This summary of the invention does not necessarily describe all
features of the invention.
Other aspects, features and advantages of the present invention will become
apparent to those of
ordinary skill in the art upon review of the following description of specific
embodiments of the
invention.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0015] These and other features of the invention will become more apparent
from the following
description in which reference is made to the appended drawings wherein:
[0016] Figure 1 is a visual representation of the interaction between one
example of a molecule
of interesnet, specifically melamine, and one example an analyte detecting
anaoparticle, in this
case gold nanoparticles, in the hydrogel when viewed by the naked eye;
[0017] Figure 2 is a molecular representation of one example of analyte
detecting
ananoparticles, in this case gold nanoparticles interacting and dissolving a
molecule of interest,
in this case a melamine solution. The nanoparticles are initially spread out
like a colloid, then
clump together by interacting with the melamine molecules due to a change in
pH herein;
[0018] Figure 3 is a visual representation of the dissolving of heavy metal in
one example of an
analyte detecting nanoparticle, in this case a gold nanoparticle-bonded
oligonucleotide solution.
A colour change is observed as the pH change breaks apart the
oligonucleotides, forcing them to
bond with the Lead molecules;
[0019] Figure 4 is a schematic diagram of one embodiment of the invention, the
light grey
represents the hydrogel bounded in the substrate vessel. A solution containing
a sample of the
molecule of interest, represented in a darker grey, is added to the substrate
vessel. The hydrogel
absorbs the molecule of interest and a colour change is shown as forming, said
color change
being visible to the naked eye;
[0020] Figure 5 is a visual representation of the interaction between one
exampleof a molecule
of interest, in this case melamine contaminants, and one example of an analyte
detecting
nanoparticle, in this case silver nanoparticles, in the hydrogel when viewed
by the naked eye;
[0021] Figure 6 is an illustration of one example of linker molecule coated
nanoparticles of the
present invention, namely 11-mercaptoundecanoid acid coated particles.
PREFERRED EMBODIMENTS OF THE INVENTION
[0022] A detailed description of one or more embodiments of the invention is
provided below
along with accompanying figures that illustrate the principles of the
invention. As such this
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detailed description illustrates the invention by way of example and not by
way of limitation.
The description will clearly enable one skilled in the art to make and use the
invention, and
describes several embodiments, adaptations, variations and alternatives and
uses of the invention,
including what we presently believe is the best mode for carrying out the
invention. It is to be
clearly understood that routine variations and adaptations can be made to the
invention as
described, and such variations and adaptations squarely fall within the spirit
and scope of the
invention.
[0023] In other words, the invention is described in connection with such
embodiments, but the
invention is not limited to any embodiment. The scope of the invention is
limited only by the
claims and the invention encompasses numerous alternatives, modifications and
equivalents.
Numerous specific details are set forth in the following description in order
to provide a
thorough understanding of the invention. These details are provided for the
purpose of example
and the invention may be practiced according to the claims without some or all
of these specific
details. For the purpose of clarity, technical material that is known in the
technical fields related
to the invention has not been described in detail so that the invention is not
unnecessarily
obscured. Similar reference characters denote similar elements throughout
various views
depicted in the figures.
[0024] This description of preferred embodiments is to be read in connection
with the
accompanying drawings, which are part of the entire written description of
this invention. In the
description, corresponding reference numbers are used throughout to identify
the same or
functionally similar elements.
[0025] In the present disclosure and claims, the word "comprising" and its
derivatives including
"comprises" and "comprise" include each of the stated integers but does not
exclude the
inclusion of one or more further integers. The term track and channel may be
interchanged
herein.
[0026] The term "variation" of an invention means an embodiment of the
invention, unless
expressly specified otherwise. A reference to "another embodiment" or "another
aspect" in
describing an embodiment does not imply that the referenced embodiment is
mutually exclusive
with another embodiment (e.g., an embodiment described before the referenced
embodiment),
unless expressly specified otherwise.
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[0027] The term "including" and variations thereof mean "including but not
limited to", unless
expressly specified otherwise.
[0028] The terms "a", "an" and "the" mean "one or more", unless expressly
specified otherwise.
[0029] The term "plurality" means "two or more", unless expressly specified
otherwise.
[0030] The term "herein" means "in the present application, including anything
which may be
incorporated by reference", unless expressly specified otherwise.
[0031] The term "whereby" is used herein only to precede a clause or other set
of words that
express only the intended result, objective or consequence of something that
is previously and
explicitly recited. Thus, when the term "whereby" is used in a claim, the
clause or other words
that the term "whereby" modifies do not establish specific further limitations
of the claim or
otherwise restricts the meaning or scope of the claim.
[0032] The term "e.g." and like terms mean "for example", and thus does not
limit the term or
phrase it explains. For example, in a sentence "the computer sends data (e.g.,
instructions, a data
structure) over the Internet", the term "e.g." explains that "instructions"
are an example of "data"
is that the computer may send over the Internet, and also explains that "a
data structure" is an
example of "data" that the computer may send over the Internet. However, both
"instructions"
and "a data structure" are merely examples of "data", and other things besides
"instructions" and
"a data structure" can be "data".
[0033] The term "respective" and like terms mean "taken individually". Thus if
two or more
things have "respective" characteristics, then each such thing has its own
characteristic, and these
characteristics can be different from each other but need not be. For example,
the phrase "each of
two machines has a respective function" means that the first such machine has
a function and the
second such machine has a function as well. The function of the first machine
may or may not be
the same as the function of the second machine.
[0034] The term "i.e." and like terms mean "that is", and thus limits the term
or phrase it
explains. For example, in the sentence "the computer sends data (i.e.,
instructions) over the
Internet", the term "i.e." explains that "instructions" are the "data" that
the computer sends over
the Internet.
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[0035] As used herein, "animal" means any member of the animal kingdom,
including all
mammals and most preferably humans.
[0036] As used herein "molecule of interest" means andy molecule which may be
present in a
consumable(including and environmental sample, such as a water sample) and
which it may be
of interest to detect. It includes adulterants (chemical, biological and
other), toxins, allergens,
bacteria, pathogens, pesticides, pharmaceuticals pharmaceutical intermediates
or ingredients.
[0037] As used herein "change to a configuration of the nanoparticles which is
a visible to the
human eye" means any alteration appearing within or on the hydrogel which
reflects an alteration to
the structure or conformation of the analyte detecting nanoparticles. Analyte
detecting nanoparticles
embedded within the hydrogel directly or indirectly interact with the molecule
of interest such that
upon an interaction there is produced a change to a configuration of the
nanoparticles which is a
visible to the naked eye. This change includes color, which can be utilized by
consumers without
any scientific training.
[0038] As used herein, "an analyte detecting nanoparticle, which is at least
one of the following:
directly interactable and indirectly interactable with the molecule of
interest" giving rise to a
visual cue confirming such interaction. Preferably, the visualcue provides a
means to
detect" degree of interaction" and or amount of interaction which represents
the concentration of
molecule of interest in the sample. For example intensity color from pale to
vivid may indicate a
lower to higher concentration of the molecule of interest in the sample. For
ease of use by a
consumer without the need of training.
The Problem
[0039] Routine detection of heavy metals is largely dependent on expensive
laboratory
equipment, which needs to be operated by personnel with specialized training.
These methods
include atomic absorption/ emission spectroscopy, inductively coupled plasma
mass
spectrometry, selective cold vapor atomic fluorescence spectrometry, and
electrochemical and
optical sensing devices.
[0040] PCBs, melamine and cocaine are most commonly detected using two types
of methods,
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analytical chemistry and biochemical methods. Analytical chemistry methods
include the gas
chromatography/high-resolution mass spectrometry, gas chromatography/electron
capture
detection, high- performance liquid chromatography/photodiode array, and
electrochemical
methods. All these methods require sophisticated and expensive laboratory
equipment, can be
time consuming and labor-intensive, and rely on highly trained personnel.
[0041] Biochemical methods are largely immunoassays, such as monoclonal
antibody-based
immunoassay, competitive immunoassay, and enzyme-linked immunosorbent assay
(ELISA).
While these tests are relatively simple to do and require no costly equipment,
the preparation of
the antibodies, is tedious and expensive.
[0042] Most commonly, pathogens in environmental and clinical samples are
detected and
identified through traditional culturing and light microscopy methods,
immunoassays or
polymerase-chain-reaction (PCR) based approaches. The traditional method is
inexpensive but
very slow and some bacteria (e.g. Mycobacteria) are difficult to culture.
Immunoassays are
relatively simple and inexpensive to carry out, but the antibody preparation
and storage
requirements make them less than ideal tests. Furthermore, they require large
amount of target
molecule from the pathogen. PCR-based detection methods are reasonably fast
and very
sensitive, but they do rely on expensive equipment and trained personnel. None
of these tests are
suitable, however, to be used on-site.
[0043] Common analytical assays for nitrite quantification include UV-Vis
spectrophotometry
(Griess reaction), ion chromatography, polarography, capillary
electrophoresis, gas
chromatography coupled to mass spectrometry (GC-MS) or fluorescence
spectrophotometry.
However, most of these analytical methods have shown important limitations
such as
requirement for sample pre-treatment, low detection limits, long processing
time and lack of
portability.
[0044] In recent years several nanotechnology-based assays have been developed
in various
academic and government laboratories (e.g. cocaine, mercury), and some do
involve the change
in optical properties of silver or gold nanoparticles upon aggregation. While
several molecules
have been targeted for testing, only a melamine test is available
commercially, and none have a
substrate as simple and versatile as hydrogels.
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[0045] The available (AccuAffinity, Bioo Scientific) colorimetric melamine
tests are like a dip-
stick, and operate on the lateral flow separation of aggregated (complexed
with melamine) and
non-aggregated gold nanoparticics. The test indicates the presence of melamine
in the sample by
the absence of a color signal on the band on the strip where melamine was
imprinted. This test is
sensitive, but it does depend on antibody-conjugated nanoparticles, which may
limit its shelf life.
[0046] The present invention provides simple to use, rapid and sensitive
devices, systems and
methods for quantitative detection of one or more analytes in consumables, for
example, food,
pharmaceuticals or drinking water. It is understood that the devices and
methods herein are used
to determine both the presence of analyte and also the absence of analyte
below a desired or
predetermined amount. Accordingly, it is understood that the use of the term
"detection" or
"determining the presence of' herein is meant to include both of these
aspects.
[0047] In certain embodiments, the devices are portable and in further
preferred embodiments,
the devices are handheld. In yet further embodiments, the devices are pocket-
sized and are highly
portable to allow for consumer use without prior training.
[0048] Regarding analytes, the devices and methods can be used to detect, for
example,
adulterants, toxins, allergens, pathogens, pesticides, pharmaceuticals,
pharmaceutical
intermediates or ingredients, biopolymers and biotechnology products.
[0049] With regard to toxins, the devices and methods can be used to detect,
for example,
aflatoxin (mycotoxins), amatoxin, ergotamine, fumonisin, vomitoxin, rancidity
(histamine),
dioxins and toxins from the following pathogens: anthrax, clostridium, C.
difficile (e.g., A & B),
Salmonella, E. Coil and Pseudomonas.
[0050] With regard to allergens, the devices and methods can be used to
detect, for example,
penicillium, fungi, sulfonamides, and wheat gluten as well as other human,
animal or
mammalian allergens.
[0051] With regard to pathogens, the devices and methods can be used to
detect, for example,
anthrax, clostridium, C. difficile (e.g., A & B), Salmonella, E. Coli,
Pseudomonas as well as
other human, animal or mammalian pathogens.
[0052] With regard to adulterants, the devices and methods can be used to
detect, for example,
triazine moieties. The term "triazine moiety," as used herein, refers to an
unsubstituted or
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substituted heterocyclic 6 atom ring containing three carbon atoms and three
nitrogen atoms and
all salts, tautomers and hydrates thereof Such compounds include, but are not
limited to, halogen
substituted, nitrogen substituted, or oxygen substituted triazine, or
melamine, cyanuric acid,
melamine cyanurate, ammeline, ammelide, benzoguanamine, cyanuric chloride, and
all isomers,
salts and hydrates thereof
[0053] The term "triazine moiety" also includes all tautomers of the above
compounds as
exemplified below for cyanuric acid, melamine, ammeline and ammelide. An
additional food
adulterant than can be detected by the devices and methods described herein is
glycosaminoglycan.
[0054] The present invention comprises a simple qualitative or semi-
quantitative detection
method for food- and water-borne contaminants, such as (but not limited to)
heavy metals,
persistent environmental pollutants, food additives and pathogens (through
released toxins, DNA
fragments or other pathogen-specific molecules), to be referred to as molecule
of interest.
[0055] The detection involves obtaining a small (0.1 ¨ 5 ml) sample from the
material to be
tested (preferably a liquid), contacting the sample to a hydrogel which
comprises an analyte
detecting nanoparticle, which is at least one of the following: directly
interactable and indirectly
interactable with the molecule of interest such that upon an interaction,
there is produced a
change to a configuration of the nanoparticles which is a visible to the human
eye. Preferably,
the analyte detecting nanoparticle is an activated metal nanoparticle or non-
metal nanoparticle.
Thereafter the hydrogel absorbs the sample. If the sample contains the
molecule of interest, then
this molecule will interact with the analyte detecting nanoparticles present
in the hydrogel
leading to a change in the nanoparticles' configuration/organization (for
example, its
"aggregation"). For example, change in the intermolecular distance of
nanoparticles as a result of
aggregation is accompanied by a change in their optical properties, leading to
a change in surface
plasmon resonance. This change in optical properties can be visible to the
naked eye, and is the
basis for the detection of the molecule of interest in the sample. Such
detection is visible by the
naked eye and needs no advanced scientific equipment.
[0056] In a preferred form, the specificity of the detection arises from the
activation of the
nanoparticles, a process that comprises coating the nanoparticles with linker
molecules that
selectively interact with the molecule of interest. The sensitivity of the
test is determined by a
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combination of several factors, such as (but not limited to) the size,
concentration, shape and
composition of the nanoparticles, the selectivity of the linker molecules in
the nanoparticles'
coating, pore-size and composition of the hydrogel.
Hydrogels
[0057] One aspect of the present invention is the use of a hydrogel as a
substrate into which an
analyte detecting nanoparticle is embedded. Hydrogel is a network of polymer
chains that are
water-insoluble, sometimes found as a colloidal gel in which water is the
dispersion medium.
Hydrogels are superabsorbent (they can contain over 99% water) natural or
synthetic polymers.
Hydrogels possess also a degree of flexibility very similar to natural tissue,
due to their
significant water content.
[0058] Hydrogels are three dimensional networks of hydrophilic polymers which
are crosslinked
to form water-swellablc but water insoluble structures. The term hydrogel is
to be applied to
hydrophilic polymers in a dry state (xerogel) as well as in a wet state.
Preferably as used herein,
hydrogels are in a wet state. Hydrogels can be crosslinked in a number of
ways, as described for
example in United States Patent Publication No. 20070249059. Alternatively,
hydrogels may be
crosslinked with ionic species or by incorporation of self associating
monomers resulting in
physical crosslinking or may be effectively be rendered insoluble by
incorporation into an
interpenetrating network.
[0059] Exemplary and preferred hydrogels comprise one or more of the
following: partially
hydrolyzed poly(vinyl acetate) (PVA), poly(ethylene vinyl acetate) (PEVA),
modified PEVA,
poly(4-vinylphenol), poly(styrene-co-ally1 alcohol), poly(N-vinylpyrrolidone),
poly (alkylethers)
including poly(ethylene oxide) and poly(ethylene oxide) co-polymers
poly(vinylethers),
poly(hydroxyalkylacrylates) or methacrylates or acrylamides including
hydroxyethylacrylate,
and hydroxypropyl acrylate, substituted or unsubstituted acrylamide or
methacrylamide,
including n,n-dimethylacrylamide, n-isopropylamide and other known hydrogels
including
materials such as agarose, methylcellulose, hyaluronan, and other naturally
derived polymers. A
matrix may comprise two or more hydrogels.
[0060] There are a number of means by which the hydrogel may be formed and/or
embedded
with the analyte detecting nanoparticle. Preferably, this is accomplished by
way of a technique
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known as the "breathing method" which involves exposing the gel to a solution
containing the
nanoparticles, letting it absorb as much as possible, then dehydrating a gel
and repeating the
absorption. Mostly preferably, several cycles of this are done. The amount of
nanoparticles in the
gel is controllable by the parameters of the breathing cycle and the number of
cycles. The
breathing may include dipping the hydrogel in a solution, or pouring the
solution on the
hydrogel.
Operation
[0064] There are two preferred device configurations. The first configuration
looks like an
adhesive bandage. The hydrogel is placed on a thin plastic sheet, and is
sealed with a plastic
cover that sticks to the plastic sheet, but can be peeled off just prior to
testing. The second
configuration is for testing larger samples, or when the hydrogel is expected
expand considerably
after the addition of the sample, the hydrogel is placed in flat plastic
container that is capable of
holding its expanded size. The container is covered with and easy-to-remove
plastic cover.
[0065] So, the invention further provides a hydrogel sensor system for
measuring a property of a
consumable fluid sample, comprising: a cartridge for collecting and treating a
biological fluid
sample, said cartridge comprising a vessel defining an interior space having
side walls, a bottom
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and an openable lid, said vessel comprising a hydrogel.
[0066] The materials in the device and method are well-researched and
manufacturing of many
of their variants is well established. The detection of the molecule-of
interest through a visible
colorimetric process is simple, fast and requires no specialized training or
equipment. The test is,
therefore, a field-portable, inexpensive, rapid and selective detection method
for a great number
of molecules.
[0067] The invention is suitable for the detection of many different analytes
using materials that
are either intensively researched or are already available commercially.
[0068] Nanoparticles of noble metals, particularly gold and silver, are widely
used in analytical
applications due to their unique optical properties. They offer improved
sensitivity, speed and
versatility compared to traditional methods. The selectivity of metal-
nanoparticle-based tests
arises from the analyte-nanoparticle coating-environment interactions, which
can be electrostatic,
can depend on hydrogen bonding or electron donor-acceptor interactions. By
strategically
selecting the optimal combination of these three factors, the tests can
achieve great sensitivity
and selectivity.
[0069] The production of gold and silver nanoparticles is well known. They are
affordable and
available commercially both in untreated and conjugated (including custom-
functionalized) form
from several companies, such as Nanoprobes, Nanocs, Nanopartz, NN-Labs,
Cytodiagnostics,
and 1nnovabiosciences. Consequently, the tests using these nanoparticles have
low cost and are
simple, and do not rely on complex instrumentation and trained personnel,
which makes them
ideal for many food-safety, environmental, bio-medical and clinical tests.
Detection of heavy metals
[0070] In some embodiments the disclosed method can be used for the detection
of heavy
metals, such as cadmium, lead and mercury using activated gold nanoparticles.
As shown in
Figure 6, 13.6 +/- .4 nm gold nanoparticles are coated with 11-
mercaptoundecanoid acid. The
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aqueous solution of these nanoparticles is red. Upon addition of a heavy-metal
ion, the particles
aggregate changing the plasmon absorption properties of the particles and
turning the solution
blue.
[0071] Heavy metals can be also detected by conjugating the nanoparticles to a
combination of
glutathione, dithiothreitol and cysteine. The combination of glutathione and
dithiothreitol coated
nanoparticles aggregate in the presence of Hg(II), Pb(II), Fe(II), Fe(I11),
Zn(11), Cd(11) and
As(111) (at 10 ppm). When cycteine coated nanoparticles are included in the
mix, the
nanoparticles aggregate in response to exposure to Hg(11) and As(II1) only.
Addition of the
chelating agent 2,6-pyridinedicarboxylic acid to the nanoparticles mix
containing all three
ligands renders the particles selective for all forms of arsenic (As(III) and
As(V) salts and
organic molecules containing arsenic) at very low concentrations. This
configuration of ligands
is successful in detecting arsenic in a real sample of drinking water.
[0072] In some embodiments the disclosed test is applicable for the detection
of Hg(11) ions
using a color change in distant to aggregated silver nanoparticles from yellow
to brown/red. The
underlying principle of this embodiment is that single-stranded DNA binds with
high affinity to
unmodified silver nanoparticles, while double stranded DNA does not. Mercury
ions are known
to selectively bind between two thymidine bases in DNA, thereby creating a
double strand. DNA
strands, engineered to contain thymidines in strategic places, fold into
hairpin loops in the
presence of Hg(II) ions, loses their ability to bind silver nanoparticles and
allow them to
aggregate (in the presence of salt) allowing the visible detection of mercury
ions. Such an assay
is able to detect a linear change in Hg(II) concentration in the 25-500 nM
range. Hybridization of
DNA strands is a temperature-sensitive process and it is possible to engineer
DNA strands that
form duplexes in the presence of mercury ions at the desired temperature (e.g.
room
temperature).
Detection of PCBs
[0073] In some embodiments, the disclosed detection method can be suitable to
indicate the
presence of PCBs in aqueous environmental samples. Selective detection of
3,3',4,4'-
tetrachlorobiphenyl (PCB77, one of the more toxic forms of PCBs) can be
achieved using single-
stranded DNA aptamers conjugated to gold nanoparticles.
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[00741 Detection of PCBs using the disclosed invention involves the following
process. DNA
aptamers isolated through systematic enrichment and their complementary
strands are
immobilized on gold nanoparticles, mixed in a solution, added to the hydrogel
that is only
partially hydrated. The two complementary DNA strands on the nanoparticles
form a double
helix bringing the nanoparticles close to each other, a condition under which
the optical
properties of the particles render the solution blue. Upon addition of an
aqueous sample, the gel
expands. If the sample contains no PCBs, then the gel remains blue because the
DNA double
helices remain intact. If the sample contains PCBs, the aptamcrs will bind the
PCBs for which
they were selected. This leads to the separation of the DNA double strands,
and the separation of
to nanoparticles. As the distance between nanoparticles increases, the
solution turns red. This
allows consumers lacking a scientific background to contact the supplier if
there is an unhealthy
amount.
Detection of melamine
[0075] Within particular regions these standards are rigorously maintained,
while in other
locations the controls are lacking and often ignored. This creates a risk to
people and pets not
only within the region producing the food products, but also to any location
that imports these
food products for consumption.
[0076] Emerging food exporting markets such as China and India among others
value low cost
manufacturing and have been found to use substitute low cost filler
ingredients to further reduce
cost and increase profits. In processed foods the ability to detect this
practice is often costly and,
in some parts of the world, is currently unattainable.
[0077] In dairy products and other food products a composition of melamine is
added to the food
product. Melamine enables the food product to mimic high protein pure dairy
products, but in
fact has no nutritional value. This practice was discovered in baby formula,
white chocolates and
dairy creamers all originally produced in China. Naturally diluting infant
formula in and of itself
is simply wrong in that infants are most in need of proper nutrition and the
harm caused is
irreparable. The problem is more severe in that the filler product, melamine,
combines easily and
quickly with acids in particular cyanuric acid (2,4,6 trihydroxy-1,3,5
triazine) to create a lethal
toxin unfit for consumption. This by-product forms in aged or less refined
melamine and when
consumed the kidney rapidly produces kidney stones and can lead to kidney
failure
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[0078] In some embodiments the disclosed invention is suitable for the
detection of melamine at
the ppb level. Gold nanoparticles arc funetionalized with 2,4,6-
trinitrobenzenesulfonic acid
(TNBS). The electron rich amine groups of melamine have a strong charge-
transfer interaction
with the electron deficient aromatic part of TNBS on the surface of the gold
nanoparticles. This
interaction decreases the interparticle distance between the nanoparticles and
causes aggregation
resulting in the red-to-blue color change in the solution. The interaction
between melamine and
TNBS is very rapid (1 min) and very sensitive, allowing detection of melamine
at 5ppb, which is
far below all safety limits.
[0079] Alternative gold nanoparticles ligands for the detection of melamine
are uracil-5-
carboxylic acid and 1-(2-mercaptoethyl)-1,3,5-triazinane-2,4,6-trione, which
can detect
melamine in the 1 ppm, and 2.5 ppb, respectively.
[0080] In some embodiments, melamine can also be detected using dopamine-
functionalized
silver nanoparticles. Dopamine, though its two hydroxyl groups is able to
reduce metal ions into
metal nanoparticles while at the same time it binds to the surface of the
particles producing an
adherent polydopamine coating and stabilizing the silver nanoparticles.
Addition of melamine to
dopamine-functionalized silver nanoparticles induces the aggregation of the
particles as the
dopamine and melamine react in an alkalic mileau through Michael addition and
Schiff base
reactions. The aggregation leads to a visible color change from yellow to
brown. The color
change is in correlation with the concentration of melamine in the 0.08-10.0
mM (0.01-1.26
ppm) range, which is below the allowable limit for melamine in milk products
in most countries.
This allows consumers lacking a scientific background to contact the supplier
if there is is an
unhealthy amount.
[0081] P-nitroaniline-modified silver nanoparticles also exhibit sensitivity
and selectivity for
melamine. Melamine induces the aggregation of these silver nanoparticles
through an electron
donor¨acceptor interaction between melamine and p-nitroaniline at the
nanoparticle interface.
The aggregation leans to a color change in the solution from yellow to blue.
This method is
suitable to detect melamine in infant formula at 0.1 ppm.
[0082] In some embodiments of the invention it can serve to detect melamine
without the need
to functionalize the nanoparticles. Small (5nm), citrate-capped, gold
nanoparticles show
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selectivity for melamine through electrostatic interactions in the absence of
particle activation.
As the citrate capping arises from the gold-nanoparticles
production/manufacturing process, a
hydrogel-based test can therefore be prepared without the need to pretreat the
gold nanoparticles.
The testing procedure involves removing proteins and lipids from liquid milk
samples or
solutions of solid milk products by the addition of trichloroacetic acid. The
precipitates are
removed by simple filtration. The remaining contaminants (e.g. lactose,
glucose, salts) are not
removed but bind to the nanoparticles at an order of a magnitude higher
concentration than
melamine. The procedure takes only a few minutes and allows the detection of
melamine in the
1-120 mg/L range through a colorimetric reaction that is visible to the naked
eye and is usable by
those without a extensive scientific background.
[0083] Citrate treated silver nanoparticles are also suitable for the
detection of melamine below
the safety limit. Similar to the gold nanoparticles, the citrate coating
renders the silver
nanoparticles negatively charged and evenly dispersed in solution. Melamine's
positive ¨NH2
group attracts these negative charges and causes the aggregation of the
particles with resultant
color change from yellow to red (Figure 5).
Detection of pathogens
[0084] In some embodiments the disclosed invention can be applied for the
detection of Shiga
toxins (Stx), released by Escherichia coil 0157:H7 and Shigella dysentriae.
These toxins
recognize cell surface glycolipids in lipid rafts. Glycan encapsulated gold
nanoparticles (GNP)
are excellent substrate on which to display multivalent glycans similar to the
glycocalyx
structures covering the surface of cells. Variants of Pk trisaccharide glycans
show selectivity to
Stxl and Stx2, two distinct forms of the Shiga toxins. The specificity of the
recognition is
sufficient to suggest the feasibility of using selected glycan ligands as
nanoparticles coating in
the disclosed invention.
[0085] The glycol ipid globotriaosylcer-amide (globotriose antigen)
specifically recognizes the B
subunit of Shiga toxins and is easily bound to gold nanoparticles. As a result
is suitable to be
used as a ligand in an assay based on the disclosed invention.
[0086] Pathogens can also be detected through hybridization of specific
oligonucleotides to their
genomic DNA. Extraction of bacterial DNA is not necessary if the bacterial
cells are subjected to
treatment with carvacrol, the major phenolic component of the essential oils
of oregano and
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thyme that can break bacterial cells in a few minutes.
[0087] In some embodiments the disclosed invention is suitable to detect
specific genomic DNA
sequences of pathogenic bacteria. A DNA probe, homologous in all mycobacterial
species, is
linked to gold nanoparticles. In an acidic solution the probe-linked
nanoparticles aggregate,
resulting the visible color change from red to blue. If a complementary DNA is
added to the
solution, it prevents the acid-induced aggregation of nanoparticles, and the
solution remains red.
Therefore, if a sample contains the DNA sequence for which the probe has
specificity, the
lo solution will not turn blue. The Ll (toxin gene) of Escherichia coli can
also be detected using a
similar strategy.
Detection of cocaine
[0088] In some embodiments the disclosed pre-assembled and portable test kit
is suitable for the
rapid colorimetric detection of 50 1tM of cocaine in aqueous solutions. The
detection involves
anti-cocaine single stranded DNA aptamers, which were added to unmodified gold
nanoparticles
(3 nm, 3.5nM) and NaC1 . In the absence of cocaine, the aptamers bind to the
nanoparticles
keeping them isolated from one another and resulting in a red solution. The
affinity of the
optamers for cocaine is very high, and the presence of cocaine the aptamers
bind the aptamers,
removing them from the nanoparticles, which lead to the solution turning blue.
Detection of cysteine and homocystein
In some embodiments the disclosed method is able to selectively detect
cysteine in the
concentration range of 0.1-5 p,M and differentiate it from other amino acids.
The detection relies
on cysteine's ability to displace single stranded DNA strands from the surface
of gold
nanoparticles by forming a strong bond between its thiol group and gold. Upon
addition of
[0089] NaCI, the cysteine-bound nanoparticles aggregate turning the previously
red solution
blue. As cysteine is the only amino acid with a thiol group, no other amino
acid is able to induce
the same effect.
[0090] An alternative to this method is one where Cu2+ are used to induce the
aggregation of
gold nanoparticles, which allows the detection of cysteine in the 10 nM range.
[0091] Flourosurfactant-capped gold nanoparticles also aggregate in the
presence of cysteine, as
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well as in the presence of homocysteine. In solutions of low ionic strength
large (-40nm)
nanoparticles preferentially aggregate in the presence of homocysteine,
therefore allowing the
selective detection of these two related molecules. This assay was successful
in detecting and
distinguishing cysteine and homocysteine in human urine samples.
Detection of nitrates
[0092] In some embodiments of the disclosed invention, aimed at the detection
of nitrites and
nitrates, two types of gold nanoparticles are functionalized. one with
541,21dithiolan-3-yl-
pentanoic acid [2-(4-amino-phenyl)ethyl]amide and the second with
541,2]dithiolan-3-yl-
pentanoic acid [2-(naphthalene-1-ylamino)et- hyl[amide and both are further
cofunctionalized
with 11-mercapto-undecy1)- trimethyl-ammonium to increase solubility. When
dispersed in
aqueous solution the nanoparticles are red. In the presence of nitrite ion
under acidic conditions,
however, the amine groups on the first nanoparticles are converted into a
diazonium salt, which
then couples with the ligand on the second nanoparticle to form covalently
interconnected
nanoparticles. This reaction causes the formation of crosslinked particle
networks which
precipitate rapidly, causing the solution to change from red to colorless. The
assay is able to
detect nitrite in drinking water at concentration above 21.7 uM, which is the
allowable limit set
by the US Environmental Protection Agency. The test can be extended to
nitrates by an
additional enzymatic reaction, the reduction of nitrates with nitrate
reductase in the sample, a
reaction which produces nitrites.
[0093] While the forms of node/apparatus, method and system described herein
constitute
preferred embodiments of this invention, it is to be understood that the
invention is not limited to
these precise forms. As will be apparent to those skilled in the art, the
various embodiments
described above can be combined to provide further embodiments. Aspects of the
present
systems, methods and nodes (including specific components thereof) can be
modified, if
necessary, to best employ the systems, methods, nodes and components and
concepts of the
invention. These aspects are considered fully within the scope of the
invention as claimed. For
example, the various methods described above may omit some acts, include other
acts, and/or
execute acts in a different order than set out in the illustrated embodiments.
[0094] Further, in the methods taught herein, the various acts may be
performed in a different
order than that illustrated and described. Additionally, the methods can omit
some acts, and/or
employ additional acts.
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[0095] These and other changes can be made to the present systems, methods and
articles in light
of the above description. In general, in the following claims, the terms used
should not be
construed to limit the invention to the specific embodiments disclosed in the
specification and
the claims, but should be construed to include all possible embodiments along
with the full scope
of equivalents to which such claims are entitled. Accordingly, the invention
is not limited by the
disclosure, but instead its scope is to be determined entirely by the
following claims.
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