CA2812312A1 - 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 - Google Patents

Abstract

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 sample comprises a hydrogel comprising 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

Description

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
DM_VAN/277754-00009/7948221.1 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 feed 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 day, 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 OrganizationA
1 Ang et al. (1 September 2009). 'The risk of melamine-induced nephrolithiasis in youno children starts at a lower intake level than [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 sample which system comprises a hydrogel comprising 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.

[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 comprises exposing the consumable sample to a hydrogel, said hydrogel comprising an analyte detecting nanoparticle; 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 human eye.

[0011] The present invention provides, in another aspect, a hydrogel embedded with an analyte detecting nanoparticle, such analyte detecting nanoparticle capable of interacting with a molecule recommended by the WHO". Pediatric Neohrolooy. Retrieved 2009-10-of interest and of changing by such interaction in a way that creates a visual cue to the human eye.

[0012] The present invention provides, in another aspect, a home test kit for the detection of molecule of interest in consumable food and beverage products, which is usable by a consumer without requiring any expensive equipment and/or special laboratory training, said kit comprising 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.

[0013] 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.

[0014] 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.

[0015] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0001] 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:

[0002] Figure la is a top plan view of a testing device of the present invention;
[0003] Figure lb is a top plan view of a testing device of the present invention [0004] Figure 2a is a side view of a testing device of the present invention;
[0005] Figure 2a is a side view of a testing device of the present invention [0006] Figure 3 is an chemical illustration of 11-mercaptoundecanoid acid coated nanoparticles;
[0007] Figure 4 is a set of two photographs (a and b) of the Colorimetric changes of GSH/DTT/Cys- modified gold nanoparticles in the presence of PDCA upon addition of a) various metal ions (5 ppb) and b) different concentrations of As11;2 "081 Figure 5 is a depiction of a sensitive and selective colorimetric detection method for to mercury(II) based on the conformation change of mercury-specific oligonucleotides (MSO) from random coil structure to hairpin structure upon the addition of Hg2+ and the phenomenon of salt-induced unmodified silver nanoparticles (AgNPs) aggregation;3 [0009] Figure 6 illustrates that DNA aptamers isolated through systematic enrichment and their complementary strands may be immobilized on gold nanoparticles, mixed in a solution, added to the hydrogel that is only partially hydrated;
[0010] Figure 7 is a schematic representation of the Ag NPs colorimetric mechanism for melamine detection; 4 [0011] Figure 8 illustrates the visual detection of cocaine using two engineered short pieces of cocaine-specific aptamers and unmodified AuNPs;5 and [0012] Figure 9 illustrates that, in the presence of cysteine, cysteine binds to the AuNPs surface via Au-S bond, spontaneously driving ssDNA molecules away from the nanoparticles, which 2 Kalluri, JR et al., (2009). Use of Gold Nanoparticles in a Simple Colorimetric and Ultrasensitive Dynamic Light Scattering Assay: Selective Detection of Arsenic in Groundwater. Angewandte Chemie International Edition, 48(51), 9668-9671 3 Wang, Y, Yang F, Yang, X (2010). Colorimetric Detection of Mercury(II) Ion Using Unmodified Silver Nanoparticles and Mercury-Specific Oligonucleotides. ACS Appl. Mater. Interfaces, 2(2), pp 339-4 Ping, H, Zhang, M, Li, H, Li, S, Chen, Q, Sun, C, Zhang, T (2012). Visual detection of melamine in raw milk by label-free silver nanoparticles. Food Control 23,191-197 5 Zhang J, Wang LH, Pan D, et al. (2008). Visual cocaine detection with gold nanoparticles and rationally engineered aptamer structures. Small, 4: 1196-1200 leads to the AuNPs aggregation under the condition of NaCl introduction, and the corresponding color change from red to blue.6 PREFERRED EMBODIMENTS OF THE INVENTION
[0013] 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 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.
[0014] 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.
[0015] 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.
6 Chen Z, Luo S L, Liu C B, et al. (2009). Simple and sensitive colorimetric detection of cysteine based on ssDNA-stabilized gold nanoparticles. Anal Bioanal Chem, 395: 489-494

[0016] 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.

[0017] 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.

[0018] The term "including" and variations thereof mean "including but not limited to", unless expressly specified otherwise.

[0019] The terms "a", "an" and "the" mean "one or more", unless expressly specified otherwise.

[0020] The term "plurality" means "two or more", unless expressly specified otherwise.

[0021] The term "herein" means "in the present application, including anything which may be incorporated by reference", unless expressly specified otherwise.

[0022] 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.

[0023] 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"
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".

[0024] 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.

[0025] 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.

[0026] As used herein, "animal" means any member of the animal kingdom, including all mammals and most preferably humans.

[0027] As used herein "molecule of interest" means any molecule which may be present in a consumable (including an environment sample, such as a water sample) and which it maybe of interest to detect. It includes adulterants (chemical, biological and other), toxins, allergens, bacteria, pathogens, pesticides, pharmaceuticals, pharmaceutical intermediates or ingredients, biopolymers and biotechnology products.

[0028] 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 human eye. This change includes color changes.

[0029] As used herein, "an analyte detecting nanoparticle" means any molecule or compound which is embedded in the hydrogel and which either directly interacts or indirectly interacts with the molecule of interest" giving rise to a visual cue confirming such interaction.
Preferbly, the visual cue provides a means to detect "degree of interaction" and/or amount of interaction which represents the conentration of molecule on interest in the sample. For example, intensity color from pale to vivid may indicate a lower to higher concentration of the molecule on interest in the sample.
The Problem

[0030] 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.

[0031] PCBs, melamine and cocaine are most commonly detected using two types of methods, 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The available (AccuAffinity, Bioo Scientific) colorometric melamine tests are like a dip-stick, and operate on the lateral flow separation of aggregated (complexed with melamine) and 0 non-aggregated gold nanoparticles. 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.

[0037] 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.

[0038] In certain embodiments, the devices are portable and in further preferred embodiments, the devices are hand-held. In yet further embodiments, the devices are pocket-sized and are highly portable.

[0039] 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.

[0040] 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. Coli and Pseudomonas.

[0041] With regard to allergens, the devices and methods can be used to detect, for example, penicillin, fungi, sulfonamides, and wheat gluten as well as other human, animal or mammalian allergens.

[0042] 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.

[0043] 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 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, arnmeline, ammelide, benzoguanamine, cyanuric chloride, and all isomers, salts and hydrates thereof.

[0044] 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.

[0045] 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.

[0046] 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 (also called functionalized) 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.

[0047] 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 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

[0048] 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.

[0049] Hydrogels are three dimensional networks of hydrophilic polymers which are crosslinked to form water-swellable 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, expressly incorporated by reference herein. 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.

[0050] 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 =substituted acrylamide or methacrylamide, including n,n-dimethylacrylamide, n-isopropylamide and other known hydrogels including "natural"
materials such as agarose, methylcellulose, hyaluronan, and other naturally derived polymers. A
matrix may comprise two or more hydrogels.

[0051] 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 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 the 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

[0052] There are two preferred device configurations as represented by Figures la and lb and 2a and 2b. The first preferred configuration of Figures 1 a and lb looks like an adhesive bandage.
Device is generally indicated at 10 and comprises a plastic sheet/substrate 11 and thin plastic cover 12 covering hydrogel 14 in an unopened state. Hydrogel 14 (comprising the analyte detecting nanoparticle) during manufacture is placed on substrate (may be a plastic sheet, preferably) and the thin plastic cover is sealed over top. The thin plastic cover sticks to the plastic sheet, but can be peeled off just prior to testing. Sample 16 is dropped onto the surface of hydrogel 14 and is absorbed yielding sample results by color change.

[0053] The second configuration is for testing larger samples, or when the hydrogel is expected expand considerably after the addition of the particular sample. In this case it is preferred that the hydrogel (comprising the analyte detecting nanoparticle) 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. This configuration is represented by Figures 2a and 2b. Device is generally indicated at 18 and comprises plastic container 20, cover 22 and container-enclosed hydrogel 24. Sample 26 is dropped onto the surface of hydrogel 24 and is absorbed therein. Hydrogel 24 expands into expanded gel 28, yielding sample results by color change.

[0054] So, the invention further provides a hydrogel sensor system for measuring a property of a consumable fluid sample, comprising: a cartridge for collecting and testing a fluid sample, said cartridge comprising a vessel defining an interior space having side walls, a bottom and a openable lid, said vessel comprising a hydrogel and wherein said hydrogel comprises an analyte detecting nanoparticle.

[0055] 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 colorometric 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.

[0056] The invention is suitable for the detection of many different molecules using materials that are either intensively researched or are already available commercially.

[0057] 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.

[0058] In a preferred form, the analyte detecting nanoparticle comprising activated metals or non-activated metals, including gold and silver.

[0059] The production of gold and silver nanoparticles is well known 7' 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 Innovabiosciences. 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

[0060] 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 3, 13.6 +/- .4 nm gold nanoparticles are coated with 11-mercaptoundecanoid acid.
The 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.

[0061] 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(ll), Fe(III), Zn(II), Cd(1) and As(lll) (at 10 ppm). When cycteine coated nanoparticles are included in the mix, the nanoparticles aggregate in response to exposure to Hg(ll) and As(M) 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 (Figure 4). This configuration of ligands is successful in detecting arsenic in a real sample of drinking water (Bangladeshi well water).

[0062] In some embodiments the disclosed test is applicable for the detection of Hg(II) 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(ll) ions, loose 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 (Figure 5).
7 Liz-Marzan, L.M. (2004). Nanometals: formation and color. Materials Today, Vol. 7, No. 2,. pp. 26-31 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).

[0063] Figure 5 represents a sensitive and selective colorimetric detection method for mercury(II). It is based on the conformation change of mercury-specific oligonucleotides (MSO) from random coil structure to hairpin structure upon the addition of Hg2+ and the phenomenon of salt-induced unmodified silver nanoparticles (AgNPs) aggregation. The calibration curve showed that the net absorption ratio value at 395 and 570 nm increased linearly over the Hg2+
concentration range of 25-500 nM with a limit of detection of 17 nM. The other environmentally relevant metal ions did not interfere with the determination of Hg2 .
Detection of PCBs

[0064] 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.

[0065] Detection of PCBs using the disclosed invention involves the following process (Figure 6). 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 aptamers will bind the PCBs for which they were selected. This leads to the separation of the DNA double strands, and the separation of nanoparticles. As the distance between nanoparticles increases, the solution turns red.
Detection of melamine

[0066] 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.

[0067] 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.

[0068] 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.

[0069] In some embodiments the disclosed invention is suitable for the detection of melamine at the ppb level. Gold nanoparticles are functionalized 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.

[0070] 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.

[0071] In some embodiments, melamine can also be detected using dopamine-functionalized silver nanoparticles. Dopamine, through 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 alkaline 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.

[0072] P-nitroaniline-modified silver nanoparticles also exhibit sensitivity and selectivity for to 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 leads to a color change in the solution from yellow to blue.
This method is suitable to delect melamine in infant formula at 0.1 ppm.

[0073] 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 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 colorometric reaction that is visible to the naked eye.

[0074] 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 7).
Detection ofpathogens

[0075] In some embodiments the disclosed invention can be applied for the detection of Shiga toxins (Stx), released by Escherichia coli 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. Varianst of Pk trisaccharide glycans show selectivity to St/a and Stx2, two distinct forms of the Shiga toxins.

[0076] The glycolipid 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.

[0077] 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 thyme that can break bacterial cells in a few minutes.

[0078] 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 solution will not turn blue. The Ll (toxin gene) of Escherichia coli can also be detected using a similar strategy.
Detection of cocaine

[0079] In some embodiments the disclosed test is suitable for the rapid colorimetric detection of 50 p.M 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 (Figure 8). 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 aptamers for cocaine is very high, and in the presence of cocaine the aptamers bind the cocaine molecules, removing them from the nanoparticles, which leads to the solution to turn blue (Figure 8).

Detection of cysteine and homocysteine

[0080] In some embodiments the disclosed method is able to selectively detect cysteine in the concentration range of 0.1-51.tM and differentiate it from other amino acids (Figure 9). 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 thoiol group and gold.
Upon addition of NaC1, 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.

[0081] 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.

[0082] Fluorosurfactant-capped gold nanoparticles also aggregate in the presence of cysteine, as well as in the presence of homocystein. 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 essay was successful in detecting and distinguishing cysteine and homocysteine in human urine samples.
Detection of nitrites and nitrates

[0083] In some embodiments of the disclosed invention, aimed at the detection of nitrites and nitrates, two types of gold nanoparticles are functionalized, one with 5-[1,2]dithiolan-3-yl-pentanoic acid [2-(4-amino-phenyl)ethyl]amide and the second with 5-[1,2]dithiolan-3-yl-pentanoic acid [2-(naphthalene-1-ylamino)et- hyliamide and both are further cofunctionalized with 11-mercapto-undeey1)- 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 I'M, 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 nitrites8

[0084] 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.

[0085] 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.

[0086] 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.
8 Weston LD, Min SH, Lee, J-S, Mirkin, CA (2009). Colorimetric Nitrite and Nitrate Detection with Gold Nanoparticle Probes and Kinetic End Points. J Am Chem Soc, 131, 6362-6363

Claims (4)

WHAT IS CLAIMED IS:

1. 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 sample which system comprises a hydrogel comprising 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.

2. A method of detecting at least one of a presence of, absence of, or concentration of a molecule of interest in a consumable sample comprises exposing the consumable sample to a hydrogel, said hydrogel comprising an analyte detecting nanoparticle;
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 human eye.

3. A hydrogel embedded with an analyte detecting nanoparticle, such analyte detecting nanoparticle capable of interacting with a molecule of interest and of changing by such interaction in a way that creates a visual cue to the human eye.

4. A home test kit for the detection of molecule of interest in consumable food and beverage products, and environmental samples which test kit is usable by a consumer without requiring any expensive equipment and/or special laboratory training, said kit comprising 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.