Physical Entrapment of a Color-Changing Indicator to a Substrate

Field The present disclosure relates to physical entrapment of a color-changing indicator to a substrate.

Background

Color changing indicators are used to provide a visually discernable indication as to the presence of a particular stimulus. The color changing indicators may be directly exposed to a surface containing an associated stimulus. However, with direct exposure to a surface, the color changing indicator that has changed color on the surface may remain on the surface and cause staining damage to the surface.

Summary The present disclosure relates to a reactive article for delivering a color changing indicator to a surface to detect the presence of a stimulus on the surface. In one embodiment, the reactive article comprises a substrate, a color changing indicator that produces a color change in response to a stimulus, and a delivery mechanism containing the color changing indicator. The delivery mechanism is secured to the substrate. The stimulus penetrates the delivery mechanism to produce a visually discernable color change of the color changing indicator.

Detailed Description

Securing color changing indicators to a substrate may have the adverse consequence of limiting or completely preventing the ability of the color changing indicator to produce a color change in response to an associated stimulus. There is a need for a delivery mechanism for exposing a color changing indicator to a surface to achieve a color change while maintaining the ability of the color changing indicator to produce a color change in the presence of a stimulus. The present disclosure relates to physical entrapment of a color-changing indicator to a substrate by including a delivery mechanism for the color changing indicator. The color changing indicator produces a

color change in response to a stimulus in that the stimulus is able to penetrate the delivery mechanism to react with the color changing indicator and produce a visually discernable color change of the color changing indicator.

I. Color Changing Indicator

The color-changing indicator is an indicator that chemically reacts in the presence of a stimulus to produce a visual color-change. The stimulus may be pH, protein, amine, sugar including glucose, hemoglobin/myoglobin, bacteria, a toxin, or a microorganism to give a reaction for the particular color-changing indicator. Typically, the stimulus will be associated with a particular contaminant. For example, if the color- changing indicator responds to amino groups, then the color-changing indicator will respond to a meat-based protein. Protein is present in meat. Meat products such as beef can carry E. coli and chicken can carry Salmonella. Therefore, a color-changing indicator that responds to an amino group may indicate a meat protein is present and contaminations such as E. coli or Salmonella may be present.

The color-changing indicator will give a visually discernable color change. In one embodiment, the color-changing indicator will give a visually discernable color change under room temperature conditions. It may be desirable to achieve a visually discernable color change within 15 minutes and further within 5 minutes. In one embodiment, it may be desirable to achieve a visually discernable color change within 60 seconds.

One suitable color-changing indicator is ninhydrin that chemically reacts in the presence of amino acids, amines and amino sugars to form a vivid purple product called Ruhemann's Purple. Therefore, ninhydrin can detect a protein by reacting to the amino group of the protein. Ninhydrin is commercially available in a hydrate formation as triketohydrindane hydrate, 2,2-dihydroxy-l,3-indandione. At room temperature, the hydrate is a stable, pale yellow, slightly hygroscopic crystalline powder. In certain solutions, the ketone 1,2,3-Indantrione may be present in less than 3%. The reaction from ninhydrin to the conjugate Ruhemann's Purple is shown below:

Ninhydrin Ruhemann's Purple

Other color-changing indicators that may be suitable include a bicinchoninic acid (BCA) assay. For BCA assay, copper sulfates (CuSO ₄ ) reacts with protein at basic conditions to reduce the Cu ²⁺ ion to Cu ⁺ . Then, the Cu ⁺ ion complexes with BCA to form a purple colored complex. The Bradford Assay (Coomassie Brilliant Blue G-250), Lowry Assay, Biuret Assay, all capable of giving color changes in the presence of a protein, may be used. Further, hemoglobin and glucose detection systems may be used. One hemoglobin system is 3,3'5,5'-tetramethylbenzidine (TMB) and cumen hydroperoxide in a buffer solution. Another hemoglobin system is 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH), 3-(dimethylamino)benzoic acid (DMAB), and hydrogen peroxide (H ₂ O ₂ ) in a buffer solution. Other hemoglobin system are benzidine, o-tolidine, o-toluidine, and o-dianisidine each in a peroxide system in a buffer solution. The MBTH/DMAB hemoglobin detection system can be modified by adding glucose oxidase and peroxidase such as horseradish to be used to detect glucose. Other systems can be used to detect glucose, such as Kl/glucose oxidase/peroxidase.

II. Substrate

A substrate is a solid support structure that provides a supporting structure for carrying, transporting, and exposing the color-changing indicator to a surface. The substrate can be made to comprise materials that include: films, paper, woven or knitted materials, nonwoven materials, membranes, foams, or sponges or a variety of combinations therefore. The substrate may be made from a natural material or a polymer based material, or a combination of natural or polymer based materials. In some embodiments, the substrate may be dry loaded with soap, surfactant, perfumes, antibacterial, antifungal, antimicrobial, or a disinfectant. In some

embodiments, the substrate may be wet loaded. In a wet state, the substrate may be saturated with solutions of water, alcohols, detergents, surfactants, antibacterial, antifungal, antimicrobial, or disinfectants, or combinations thereof. In either a dry or wet loaded substrate, the additive should not adversely affect stability of the color-changing indicator or the color-changing indicator's ability to give a color change in the presence of the stimulus. Disinfectants may be particularly suitable for incorporation into a substrate intended for cleaning purposes. Common surface disinfectants comprise biocides such as alcohols, biguanides, cationic surfactants, and halogen or halogen containing compounds. Suitable alcohols include ethanol and isopropyl alcohol (IPA) 70% in water [IPA/H ₂ O (70/30), EtOH/H ₂ O (70/30)] . Suitable biguanides

(chlorhexidine) include polyhexamethylene biguanide, p-chlorophenyl biguanide, and A- chlorobenzhydryl biguanide. Commercially available biguanides are Nolvasan® available from Wyeth of Fort Dodge, IA and ChlorhexiDerm® Disinfectant available from DVM Pharmaceuticals of USA. Examples of cationic surfactant (Quaternary Ammonium Compounds, Quats) include Parvosol® available from Hess & Clark of

Randolph, WI, Roccal-D® Plus available from Pfizer of New York, NY, Unicide™ 256 available from Brulin & Coompany Inc. of Indianapolis, IN, and benzalkonium chloride. Typical halogen or halogen containing compounds are either chlorine or iodine based. One suitable substrate is a nonwoven web of fibers. The fibers can be synthetic polymer, natural, or a combination of synthetic or natural. In one embodiment, synthetic polymers include thermoplastic material, such as, but not limited to, polyesters, polyamides, polyimides, nylon, polyolefms (e.g., polypropylene and polyethylene), poly(ethylene vinyl alcohol) copolymer (PEVOH), poly (propylene vinyl alcohol) copolymer (PPVOH), polylactic acid (PLA), or combinations thereof. In another embodiment, the synthetic polymer of the fiber comprises regenerated cellulose, including rayon. In one embodiment natural fibers include plant based fibers such as, but not limited to, cotton, wool, linen, hemp, bamboo, soybean. Multilayer or multicomponent fibers may be used. It is understood that a variety of fiber length, diameters, sizes, shapes may be used. The fibers can have a variety of sizes and lengths. The fibers can be made in to a woven, knitted, or nonwoven article. To make a nonwoven web from fibers, a variety of processes are known including carding,

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garneting, airlaying, spunbond, wet-laying, melt blowing, stitchbonding. Further processing of a nonwoven may be necessary to add properties such as strength, durability, and texture. Examples of further processing include calendering, hydroentangling, needletacking, resin bonding, thermobonding, ultrasonic welding, embossing, and laminating.

The substrate can have any size, shape, or rigidity depending on the end use needs. Coatings of materials such as resins, surfactants, detergents, which may be included into or onto the substrate. Abrasive particles may be placed over or within the article with incorporation into resins or incorporation into a surfactant. The coatings should be applied in a way so as not to inhibit the ability of the color-changing indicator to give a color response. For example, resin may be spot coated to specified areas of the articles and not to the entire article. The article may be a layered product comprising various layers of different combinations of nonwoven, woven or knitted materials, film, foam, sponge, and various combinations thereof. If layered, the layers may be laminated, stitched, needlepunched or otherwise bonded to secure the layers together.

III. Delivery Mechanism

The delivery mechanism contains the color changing indicator. The delivery mechanism is secured to the substrate to form a reactive article. Because the delivery mechanism contains the color changing indicator, indirectly the color changing indicator is physically entrapped onto the substrate. The delivery mechanism is such that the stimulus, that is capable of producing a response with the color changing indicator, can penetrate the delivery mechanism. Further, the delivery mechanism is such that upon reaction of the color changing indicator with the stimulus, a visually discernable color change of the color changing indicator is apparent. The delivery mechanism should not inhibit that ability of the user to detect the color change of the color changing indicator upon contacting the associated stimulus.

A. Hydrogel In one embodiment, the delivery mechanism may be a hydrogel. A "hydrogel" refers to a polymeric material that is hydrophilic and that is either swollen or capable of

being swollen with a polar solvent. The polymeric material typically swells but does not dissolve when contacted with the polar solvent. That is, the hydrogel is insoluble in the polar solvent.

The color changing indicator is dispersed into the hydrogel such that the hydrogel physically entraps the color changing indicator. In one embodiment, the stimulus is capable of penetrating into the hydrogel to react with the color changing indicator. In this embodiment, the color change is contained within the hydrogel. In another embodiment, the color changing indicator contained within the hydrogel passes out of the hydrogel to react with the stimulus. Without being limited to any particular theory, it is believed that the kinetics of the reaction between the color changing indicator and the stimulus determines whether the color change occurs within the hydrogel. Due to the porous matrix of the hydrogel, the hydrogel is capable of being loaded with a relatively high concentration of the color changing indicator.

The hydrogel can be used independently to be exposed to a stimulus. In another embodiment, the hydrogel can be coated or printed onto a portion of the substrate in a variety of shapes, configurations, or patterns. The hydrogel coated substrate can be provided as either a dry substrate or a wet substrate.

The hydrogel system chosen should be compatible with the color-changing indicator so that the color changing indicator is stable in the hydrogel and maintains its ability to produce a color change in response to a stimulus. Examples of materials that can form the hydrogel include, but are not limited to, agarose gel, polyacrylamides (PAGE), polysiloxanes, celluloses, polyethylene glycol, polyacrylates, alginate, polyvinyl alcohol, polyvinyl pyrrolidone. Also, the hydrogel system should not interfere with the user's ability to visually observe a color change of the color changing indicator.

B. Hydrogel Beads

In one embodiment, the delivery mechanism may be a hydrogel bead. US Patent application 11/759283 filed on June 7, 2007 titled "Polymeric Beads and Methods of Making Polymeric Beads," herein incorporated by reference, discloses a hydrogel bead suitable for use as the delivery mechanism of the color changing indicator.

The color changing indicator is dispersed throughout the hydrogel of the hydrogel bead. In one embodiment, the stimulus is capable of penetrating into the matrix of the hydrogel bead to react with the color changing indicator within the bead. In this embodiment, the color change is contained within the hydrogel bead. In another embodiment, the color changing indicator contained within the hydrogel passes out of the hydrogel bead to react with the stimulus.

The hydrogel system of the hydrogel bead chosen should be compatible with the color-changing indicator so that the color changing indicator is stable in the hydrogel and maintains its ability to produce a color change in response to a stimulus. Examples of materials that can form the hydrogel bead include, but are not limited to, acrylates, such as ethoxylated TMPTA (trimethyl propane triacrylate). The hydrogel beads may range in diameter size from 1 micro to 3000 microns. The hydrogel system of the hydrogel bead should not interfere with the user's ability to visually observe a color change of the color changing indicator. The hydrogel bead independently can be used to react with the stimulus. In another embodiment, the hydrogel beads can be secured to a portion of the substrate. In one embodiment, the hydrogel beads can be adhesively secured to a portion of the substrate. In another embodiment, the hydrogel bead can be entrapped or contained within a fibrous substrate or within a pocket, package, or mesh area of a substrate. The securing mechanism should not interfere with the ability of the color changing indicator's ability to produce a color change in the presence of a stimulus. Also, the securing mechanism should not interfere with the user's ability to visually observe a color change of the color changing indicator.

C. Hydrogel Fibers

In one embodiment, the delivery mechanism may be a hydrogel fiber. US Patent application 60/891260 filed on February 23, 2007 titled "Polymeric Fiber and Methods of Making," and 60/946,745 filed on June 28, 2007 titled "Polymeric Fiber and Methods of Making" both herein incorporated by reference, disclose a hydrogel fiber suitable for use as the delivery mechanism of the color changing indicator.

The color changing indicator is dispersed throughout the hydrogel of the hydrogel fiber. In one embodiment, the stimulus is capable of penetrating into the matrix of the hydrogel fiber to react with the color changing indicator within the bead. In this embodiment, the color change is contained within the hydrogel fiber. In another embodiment, the color changing indicator contained within the hydrogel passes out of the hydrogel fiber to react with the stimulus.

The hydrogel system of the hydrogel fiber chosen should be compatible with the color-changing indicator so that the color changing indicator is stable in the hydrogel and maintains its ability to produce a color change in response to a stimulus. Examples of materials that can form the hydrogel fiber include, but are not limited to, acrylates, such as ethoxylated TMPTA (trimethyl propane triacrylate). The hydrogel fibers may range in size from 10 to 100 mils and more particularly from 25 to 50 mils. The hydrogel system of the hydrogel fiber should not interfere with the user's ability to visually observe a color change of the color changing indicator. The hydrogel fiber independently can be used to react with the stimulus. In another embodiment, the hydrogel fiber can be secured to a portion of the substrate. In one embodiment, the hydrogel fiber can be adhesively secured to a portion of the substrate. In another embodiment, the hydrogel fiber can be entrapped or contained within a fibrous substrate or within a pocket, package, or mesh area of a substrate. In another embodiment, the hydrogel fiber can be incorporated in with other fibers to make a nonwoven article. In such an article an additional adhesive may be used to secure the fibers together to provide structural strength to the nonwoven. Techniques such as dry laying, carding, or air laying may be used to place the polymeric fibers onto a substrate or for blending with other fibers to form a substrate. The securing mechanism should not interfere with the ability of the color changing indicator's ability to produce a color change in the presence of a stimulus. Also, the securing mechanism should not interfere with the user's ability to visually observe a color change of the color changing indicator.

D. Polyelectrolyte Multilayer Film

In one embodiment, the delivery mechanism may be a polyelectrolyte film. US Patent application 60/913384 filed on April 23, 2007 titled "Fibrous Articles with One or More Polyelectrolyte Layers Thereon and Methods for Making the Same," herein incorporated by reference, discloses a polyelectrolyte multilayer film (PEM) suitable for use as the delivery mechanism of the color changing indicator. The PEM film is a multilayer arrangement of alternating charged layers on a substrate.

The color changing indicator is contained between one or all of the layers of the PEM film. Hydrogen bonding or ionic interaction secures the color changing indicator to the PEM layer. The stimulus diffuses through the PEM layer to react with the color changing indicator to produce a color change.

The materials chosen for the PEM layers should be compatible with the color- changing indicator so that the color changing indicator is stable in the PEM layers and maintains its ability to produce a color change in response to a stimulus. Also, PEM layers should not interfere with the user's ability to visually observe a color change of the color changing indicator.

E. Membrane Entrapping

In one embodiment the delivery mechanism may be a membrane. The color changing indicator is contained within the membrane. The stimulus, that is capable of reacting with the color changing indicator, is able to penetrate into the membrane to produce a visually discernable color change.

The membrane should be chosen so that the color changing indicator is stable within the membrane and also so that the associated stimulus is capable of penetrating the membrane to produce a visually discernable color change. The membrane chosen should be stable for storage, highly porous for high loading of the indicator and achieving rapid response. Also, the membrane should not interfere with the ability of the color changing indicator's ability to produce a color change in the presence of a stimulus. One example of a suitable membrane is an alumina membrane. Other membranes that may be useful include cellulose membranes and polymeric membranes such as polysulfone membranes.

IV. Reactive Article

A substrate with a color changing indicator physically entrapped thereon produces a reactive article. The reactive article may be a single layer structure or a multilayer structure, wherein one, some, or all of the layers contain a color changing indicator. Each layer may have the same color changing indicator or may have a different color changing indicator. It is not necessary that all layers will include a color changing indicator. The reactive article may have a variety of shapes, sizes, and constructions. The reactive article may be used in a variety of areas where a visible detection indicating the presence of a substance is desirable. The color-changing indicator chemically reacts in the presence of a stimulus to produce a visual color change. Typically, the stimulus will be associated with a particular contaminant. For example, if the color changing indicator responds to amino groups, then the color changing indicator will respond to a meat-based protein. Protein is present in meat. Meat products such as beef can carry E. coli and chicken can carry Salmonella. Therefore, a color-changing indicator that responds to an amino group may indicate a meat is present and contaminations such as E. coli or Salmonella may be present.

To use the reactive article, the reactive article is passed over a surface. If the surface is free of a stimulus capable of giving a color change with the color changing indicator, then no visual color change is apparent. Then, the user knows the surface is essentially free of that stimulus. Typically, the stimulus will be associated with a particular contaminant. Therefore, the user knows the surface is essentially free of the associated contaminant. If the surface includes the stimulus that is capable of giving a color-change with the color-changing indicator, then a visual color change will appear. The users know the surface includes the stimulus and the associated contaminant.

In one embodiment, the color changing indicator is responsive to a protein stimulus through reaction with an amine group. Therefore, a color change in the color changing indicator is indicative of protein on the surface, which may be indicative of bacteria such as E. coli or Salmonella being present on the surface.

In the embodiment where the article further includes a disinfectant, a wipe across the surface to detect a color change will also deliver a portion of the disinfectant. Therefore, upon seeing a color change some of the disinfectant will act upon the stimulus on the surface. The user may wipe the surface again with a new article to determine if the stimulus had been removed.

Although specific embodiments of this invention have been shown and described herein, it is understood that these embodiments are merely illustrative of the many possible specific arrangements that can be devised in application of the principles of the invention. Numerous and varied other arrangements can be devised in accordance with these principles by those of ordinary skill in the art without departing from the spirit and scope of the invention. Thus, the scope of the present invention should not be limited to the structures described in this application, but only by the structures described by the language of the claims and the equivalents of those structures.

Examples

1. Hydrogel system Preparation of Stimulus (Meat Juice):

A meat juice solution was prepared. Approximately 16 gram of fresh pork chop meat was extracted with 20 mL of water for 16 hours and the mixture was filtered. The total protein in the meat juice was measured according to Pierce Coomassie Plus Protein

Assay (product # 23238) available from Pierce Biotechnology Inc., Rockford, IL and ranges from approximately 17 mg/mL to 37 mg/mL.

Product List Polyvinyl alcohol) (PVA, 99+% hydrolyzed, M _w 89,000 - 98,000) available from

Aldrich Chemical Co. of Milwaukee, WI

Poly(acrylic acid) (PAA, -0.1% cross-linked, M _w -450,000) available from

Aldrich Chemical Co. of Milwaukee, WI

Ninhydrin available from Aldrich Chemical Co. of Milwaukee, WI

Preparation of PVA/PAA/Ninhydrin System

3.2 g Polyvinyl alcohol) aqueous solution (15wt%) and 0.8 g poly(acrylic acid) aqueous solution (7.5wt%) were mixed and heated at 60 ⁰ C for 1 hour. After that, ninhydrin (88 mg) was dissolved in PVA/PAA solution to make a PVA/PAA/Ninhydrin hydro-gel sensing solution. The ninhydrin concentration in solution was 2.2% by weight. The above solution was then screen printed onto non- woven substrates such as Buckeye FG 613 and Rayon/PET (60/40 or 70/30). These substrates were used later to react with meat juice.

A dry PVA/PAA/Ninhydrin coated substrate (Buckeye FG 613) was used to wipe off 3 drops of meat juice on a white tile, and it was found that the areas of the wipe with juice absorption slowly turned light purple after 15 minutes. Nevertheless, when meat juice was wiped off by a pre-wetted (IPA or EtOH) PVA/PAA/Ninhydrin coated substrate (Buckeye FG 613), it was observed that the areas of wipe with meat juice absorption turned purple color in less than 5 minutes.

2. PEM Film

Preparation of Stimulus (Meat Juice):

A meat juice solution was prepared. Approximately 16 gram of fresh pork chop meat was extracted with 20 mL of water for 16 hours and the mixture was filtered. The total protein in the meat juice was measured according to Pierce Coomassie Plus Protein

Assay (product # 23238) available from Pierce Biotechnology Inc., Rockford, IL and ranges from approximately 17 mg/mL to 37 mg/mL.

Product List Poly(diallyl dimethyl) ammonium chloride (PDDAC), MW 240,000, available from Polysciences, Inc. of Warrington, PA

Poly(styrene sulfonic acid) sodium salt (PSS), MW 70,000, available from Alfa Aesar, A Johnson Matthey Company of Ward Hill, MA

Ethylene vinyl alcohol meltblown nonwoven web made Poval PVOH of resin grade PVA 205 MB available from Kuraray America, Inc. New York, NY.

(3-acrylamidopropyl)trimethyl ammonium chloride (APTAC) 75 wt% solution in water, available from Aldrich Chemical Co. of Milwaukee, WI

Preparation of PEM Nonwoven A meltblown non-woven web made of ethylene vinyl alcohol (EVOH) was first functionalized by use of e-beam with 16wt% (3-acrylamidopropyl)trimethyl ammonium chloride (APTAC) to generate a positive charge on the surface of the web. After being functionalized, polyanion and polycation were used alternatively to form the multilayer. The polycation was poly(diallyl dimethyl) ammonium chloride (PDDAC), while the polyanion was poly(styrene sulfonic acid) sodium salt (PSS). The e-beam treated samples were soaked in 0.02 M PSS solution with pH 1.49 for 1 hour and then rinsed. Following the rinsing step, the samples were then dipped into a beaker containing a mixture of 25 mL 2wt% ninhydrin and 75 mL 0.02 M (PDDAC) solution at pH 5.38 for 20 minutes and then rinsed. This completed the first bilayer. Subsequent bilayers were created by dipping the first bilayers in a 0.02 M solution of PSS at pH about 5.40 for 20 minutes then rinsed in water. Following the rinsing step again, samples were immersed into a beaker of the PDDAC -Ninhydrin solution for 20 minutes. Following the same step, the desired number of bilayers can be achieved. In current experiment, samples with three different bilayers were made and used to investigate their reactivity towards meat juices.

Meat juices were applied onto the ninhydrin incorporated PEM non- woven web with different bilayers. No color change was observed immediately at room temperature unless heat was applied. Without heat, the purple color appeared after more that 24 hours.

3. Membrane Entrapped Ninhydrin:

Product List

Anopore® alumina vertical porous membranes available from Whatman Inc, Florham Park, NJ

Ninhydrin available from Aldrich Chemical Co. of Milwaukee, WI.

Human serum albumin (Buminate® band, 25%) available from Baxter Healthcare Corp. of Glendale, CA

Carbopol ® 1342 available from Noveon Corp, Cleveland, OH

Cytosep® hydrophilic paper (media grade 1662) available from Pall Corp, East Hills, NY

Preparation of Membrane

Anopore® alumina porous membranes (60 micron in thickness) with pore size 20 and 100 nanometers (nm) were cleaned with methanol and chloroform and dried in a desiccator overnight. The membranes were then soaked with 5wt% ninhydrin methanol solution, containing 0.1% of Carbopol® 1342 as a bounding agent, and drained. The membranes were dried and stored in a desiccator for future tests.

Cytosep® paper was used as received from the manufacturer. The paper was soaked with 5wt% ninhydrin methanol solution, containing 0.1 wt% of Carbopol® 1342 as a binding agent, and drained. The membranes were dried and stored in a desiccator for future tests.

Ten (10) wt. percent DI water solution was made by diluting the Buminate® albumin with de-ionized water. A drop of albumin solution was applied to the membrane and the membrane turned purple. The responding time was 65, 60, and 55 seconds, for ninhydrin entrapped 20 nm Anopore® membrane, 100 nm Anopore® membrane, and Cytosep® paper, respectively.