Screening method, device and kit for detecting mucosal carbohydrates and associated conditions

Abstract

To provide screening test methods, devices and kits for cancerous and precancerous conditions with improved accuracy and minimized handling procedures.SOLUTION: Provided is a screening device for rapidly testing human mucosal carbohydrate expression, comprising a test strip or membrane with pre-embedded galactose oxidase, and a container with Schiff reagent solution, where the Schiff reagent solution is not initially in contact with the test strip or membrane, and the Schiff reagent solution can be activated to contact the test strip or membrane after marker carbohydrates are oxidized by galactose oxidase. This disclosure further provides the use of the device or kit in a medical facility for an initial evaluation for cancerous and precancerous conditions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 893,484, filed August 29, 2019, and U.S. Non-Provisional Patent Application No. 16 / 937,051, filed July 23, 2020, the entire disclosures of which are incorporated herein by reference.

[0002] The present disclosure generally relates to screening test methods, devices, and kits for detecting mucosal carbohydrates and related conditions, including, but not limited to, cancerous and precancerous conditions. Specifically, the methods test for abnormal carbohydrates in mucus or body fluids using the enzyme galactose oxidase and Schiff's reagent. The present disclosure further relates to the use of the devices or kits in medical facilities for the initial evaluation of abnormal carbohydrates and related conditions. [Background technology]

[0003] Early detection and prevention remain the best strategy for combating various cancers through screening. An ideal screening test must meet certain criteria, namely, high sensitivity, specificity, and positive predictive value, cost-effectiveness, non-invasiveness, and public acceptability. Unfortunately, this effort has been hindered by the insensitivity and nonspecificity of existing screening assays for colorectal cancer, i.e., fecal occult blood tests (FOBTs), and the high cost and radiation risk of mammograms and chest x-rays for breast and lung cancer, respectively. Furthermore, detecting disease after cancer has already formed is too late, making detection at the precancerous stage even more important. Therefore, identifying specific markers and easy-to-implement, cost-effective, and accurate methods for detecting cancer and precancerous disease are crucial to effectively address this issue.

[0004] Mucins are high-molecular-weight, highly glycosylated glycoproteins. They are produced by epithelial cells in normal and malignant tissues as either secreted or membrane-bound molecules. Cancer development and progression are often accompanied by changes in the biochemical properties of mucins, including both aberrant glycosylation of the mucin core peptide and altered expression of mucin genes.

[0005] Patent Document 1 and Non-Patent Document 1 report a colon cancer screening test that can detect colon cancer using rectal mucus. A lyophilized cellulose membrane filter was pre-impregnated with an enzyme phosphate buffer solution and then moistened. The moistened cellulose membrane filter was then contacted with a Metricel membrane filter containing a mucus sample for 1 to 2 hours to react the mucus with the enzyme galactose oxidase. The membrane filter containing the mucus was then washed with distilled water for 1 minute, reacted with basic fuchsin for 15 minutes, washed with tap water for 10 minutes, and air-dried.

[0006] Patent Document 2 discloses a test kit packaged in a cardboard box in a conventional manner, which includes (a) a capped vial containing a sufficient amount of storage-stable basic fuchsin to saturate (b) twice, prepared according to the following preparation method, (b) a strip of membrane filter (Metricel membrane filter 0.46 μm, Gelman Sciences, Inc., Ann Arbor, Michigan). The kit also includes (c) a storage-stable form of galactose oxidase impregnated into a strip of membrane filter and contained in a sealed, capped bottle, in an amount sufficient to oxidize a marker carbohydrate in the sample. The kit also includes (d) periodic acid, and (e) a color chart for comparison with and interpretation of test results. Includes a route. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 4,857,457 [Patent Document 2] U.S. Patent No. 5,348,860 [Non-patent literature]

[0008] [Non-Patent Document 1] Shamsuddin et al., Human Pathology, 19: 7-10, 1988 Summary of the Invention [Problem to be solved by the invention]

[0009] However, test kits contain multiple liquid components and the testing method requires multiple steps, resulting in false negatives due to sampling or procedural errors.

[0010] Therefore, there is a need to improve screening test methods for cancerous and precancerous conditions and to develop screening test devices or kits. [Means for solving the problem]

[0011] The present disclosure relates to a screening test method, device, and kit for cancerous and precancerous conditions. Specifically, the method tests for abnormal carbohydrates in mucus or body fluids using a galactose oxidase reagent and a Schiff's reagent. The screening test method, device, and kit provide improved accuracy and minimize handling procedures. The present disclosure further relates to the use of the device or kit in a medical facility for the early evaluation of cancerous and precancerous conditions.

[0012] In one aspect, a method for screening for cancer and precancerous conditions and lesions comprises the steps of applying mucus or body fluid to a test strip or membrane pre-embedded with galactose oxidase, oxidizing marker carbohydrates in the mucus or body fluid containing marker carbohydrates by reacting with the galactose oxidase, and activating a reservoir containing a Schiff's reagent solution adjacent to the test strip or membrane to contact the test strip or membrane, wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane.

[0013] In some embodiments, the method for screening for cancerous and precancerous conditions and lesions includes one or more additional steps selected from applying water onto the test strip or membrane before applying the mucus or body fluid to the test strip or membrane, removing the mucus or body fluid by washing before activating the container containing the Schiff's reagent solution, rinsing the test strip or membrane with tap water after contacting the Schiff's reagent solution with the test strip or membrane, drying the test strip or membrane, and / or evaluating the color of the test strip or membrane.

[0014] In another aspect, a device for treating cancer and precancerous conditions and lesions includes a test strip or membrane pre-embedded with galactose oxidase and a container containing a Schiff's reagent solution, wherein the Schiff's reagent solution is initially out of contact with the test strip or membrane and can be activated to contact the test strip or membrane after the marker carbohydrate has been oxidized by galactose oxidase. The mechanism for activating the carbohydrate Schiff's reagent solution (bringing it into contact with the test strip or membrane after the marker carbohydrate has been oxidized by galactose oxidase) is not limited. In one aspect, the mechanism can be a twist valve, a breakable barrier, or the like, where the twist valve is opened or the barrier is broken before the Schiff's reagent solution contacts the test strip or membrane.

[0015] In another aspect, the present disclosure provides a test kit for cancerous and precancerous conditions, comprising a test strip or membrane pre-embedded with galactose oxidase and a container containing a Schiff's reagent solution. In one aspect, the galactose oxidase pre-embedded test strip or membrane further contains a dried culture medium that can activate the galactose oxidase upon addition of water onto the test strip or membrane. In another aspect, the pre-embedded galactose oxidase of the test strip or membrane is microencapsulated.

[0016] In another aspect, the present disclosure further relates to the use of the disclosed device or kit for screening for a cancerous or precancerous condition. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 shows the principle of the reactions involved in the kits and methods of the present invention. In Figure 1, the marker Gal-GalNAc reacts with galactose oxidase (GO) to generate two adjacent aldehydes at the C6 position, which are then conjugated with basic fuchsin, imparting a magenta color. [Figure 2] 1 shows one embodiment of the method of the claimed invention in which samples are reacted on a glass slide to identify cancerous or precancerous conditions. In samples from subjects without any cancer or precancer, the test panel is colorless, while typically a magenta color (ranging from pink to purple) indicates marker disaccharides specific for cancer and precancerous conditions and lesions. DETAILED DESCRIPTION OF THE INVENTION

[0018] The following is a detailed description presented to aid those skilled in the art in practicing the present disclosure. Those skilled in the art may make modifications and variations to the embodiments described herein without departing from the spirit or scope of the present disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated herein by reference in their entirety.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0020] Where a range of values ​​is presented, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure unless the context clearly dictates otherwise (e.g., in the case of a group having a large number of carbon atoms, where each number of carbon atoms included within a range is recited). The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, subject to any specifically excluded limit in the stated range, and are also encompassed within the disclosure. Where the stated range includes one or both of the limits, ranges excluding both of those included limits are also included in the disclosure.

[0021] All numerical values ​​in the detailed description and claims herein are modified by the stated value being "about" or "approximately" to account for experimental error and variations that would be expected by one of ordinary skill in the art.

[0022] The following terms are used to describe this disclosure: Unless otherwise defined, all technical and scientific terms used herein are understood by those of ordinary skill in the art to which this disclosure belongs. The terminology used herein has the same meaning as commonly understood in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0023] The non-quantitative articles "a" and "an" used in this specification and the appended claims are intended to be used in place of the grammatical articles "a" and "an" unless the context clearly indicates otherwise. As used herein, the term "an element" refers to one or more than one (i.e., at least one) of an object. By way of example, "an element" means one element or more than one element.

[0024] The term "and / or," as used in the specification and claims, is understood to mean "either or both" of the elements so conjunctively, i.e., elements present conjunctively in some cases, and elements present disjunctively in other cases. Multiple elements listed with "and / or" are to be construed in the same manner, i.e., "one or more" of the elements so conjunctively. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, "A and / or B," when used in conjunction with open-ended language such as "comprising," can refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements), etc.

[0025] As used in the specification and claims, "or" is understood to have the same meaning as "and / or" as defined above. For example, when separating listed items, "or" or "and / or" is to be interpreted as inclusive, i.e., the inclusion of at least one of a plurality of elements or a list of elements, but also two or more, and optionally additional unlisted items. Terms such as "only one of" or "exactly one of," or, when used in the claims, "consisting of" are also used. Only terms clearly indicating the contrary, such as "of," shall be used to refer to the exact element or list of elements. In general, the term "or" as used herein will be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") only when followed by terms of exclusion, such as "either," "one of," "only one of," or "exactly one of."

[0026] In the claims and this specification, the words "comprising" and "including ), "carrying," "having," "containing," "involving," "holding," "composed of All transitional phrases, such as "consisting of" and "consisting essentially of," shall be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0027] The phrase "at least one" as used herein and in the claims with respect to a list of one or more elements will be understood to mean at least one element selected from any one or more of the elements in the list of elements, but will not necessarily include at least one of each and every element specifically listed in the list of elements, and will not exclude any combination of elements in the list of elements. This definition also applies to any element specifically identified in the list of elements to which the phrase "at least one" refers, whether or not it relates to the specifically identified element. The term "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A (optionally including more than one) and no B (optionally including elements other than B), in another embodiment to at least one B (optionally including more than one) and no A (optionally including elements other than A), in yet another embodiment to at least one A (optionally including more than one) and at least one B (optionally including more than one) (optionally including other elements), etc.

[0028] As used herein and in the claims, the phrase "marker carbohydrate" or "marker saccharides" should be understood to mean carbohydrates that can provide information about cancer and precancer using the methods described herein, including, but not limited to, β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Galβ-(1→4)-Fuc-α-1→3-GlcNAc, Fuc-α-1→2-Galβ-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc, or Fuc-α-1→2-Gal-β-(1→4)-Fucα-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc.

[0029] As used in this specification and claims, the terms "mucous" and "body fluid" are intended to be interchangeable and are intended to be broadly interpreted as any fluid or mucus derived from the human body that contains marker carbohydrate(s).

[0030] As used in this specification and claims, the terms "embedded," "impregnated," and "pre-embedded" are intended to be interchangeable. In certain embodiments, the reagents of the present invention can be embedded directly into a membrane or test strip by binding or immobilizing the reagents on the surface of the membrane or test strip, or by loading the reagents into the pores of a porous membrane via a coating. In yet other embodiments, the reagents of the present invention are encapsulated in capsules or microcapsules that are subsequently embedded, coated, or impregnated into the reagent or test strip.

[0031] The background art and theory used in this disclosure for detecting the presence of marker carbohydrates in the mucus or body fluids of individuals being tested for cancerous or precancerous conditions is described in U.S. Patent No. 5,162,202, U.S. Patent No. 5,162,202, U.S. Patent No. 5,162,202, and ... Usefulness of Galactose Oxidase—Schiff Test in Rectal Mucus for Screening of Colorectal Malignancy ANTICANCER RESEARCH 21:1247-1256 (2001). All of these references are incorporated herein by reference. The reactions used in the methods and kits of the present invention are shown in FIG.

[0032] The present invention improves upon the assays described above with respect to minimizing false positives as a result of sampling or procedural errors, as well as for convenience.

[0033] The present disclosure provides reliable screening methods, devices and / or kits for the detection of a wide range of cancers, for example, of the rectum, colon, blood, lymph nodes, stomach, kidney, gallbladder, prostate, testicle, breast, cervix and ovary. In pre-cancerous conditions, i.e., conditions in which an individual exhibits high-risk symptoms, the individual falls into the category of "highly susceptible to subsequent cancer."

[0034] Specifically, the marker carbohydrate or sugars in the mucus or body fluid sample are present on glycoproteins. It is detected by selective oxidation of glycoproteins in mucus or body fluid samples with galactose oxidase or an equivalent oxidizing agent, which oxidizes the primary hydroxyl groups of only the galactose moieties of the sugars present to aldehyde groups. The resulting aldehyde groups can then be visualized with Schiff's reagent, e.g., basic fuchsin, which produces a magenta color.

[0035] Galactose moiety marker carbohydrates or saccharides are selectively oxidized to aldehyde sugar moieties by galactose oxidase rapidly at room temperature, e.g., in less than about 15 minutes, e.g., about 5 to 10 minutes, and even more rapidly at elevated temperatures up to the inactivation temperature of the enzyme. When using this enzyme, any ratio of enzyme to substrate and vehicle suitable for enzyme activation known in the art can be used.

[0036] The oxidized sample, with or without first removing or inactivating the galactose oxidase, is then treated with a reagent that visualizes or allows visualization of the aldehyde sugar moieties thus generated, such as fuchsin, rosaniline, magenta, or other Schiff base decolorizing dyes.

[0037] The objects, features and advantages of the present invention are achieved in one aspect by providing a rapid, reliable with respect to false negatives, and commercially viable method for detecting the presence of a precancerous or cancerous condition in humans. The present invention employs a testing method comprising obtaining a mucus or body fluid sample, assaying the sample to detect the presence therein of at least one of the marker carbohydrates β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Galβ-(1→4)-Fuc-α-1→3-GlcNAc, Fuc-α-1→2-Galβ-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc, or Fuc-α-1→2-Gal-β-(1→4)-Fucα-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, and optionally diagnosing the presence and extent of precancer or cancer based on the amount of marker carbohydrate(s) detected in the mucus or body fluid.

[0038] Storage-stable Schiff's reagent solution Dissolve 1.0 g of basic fuchsin in 200.0 mL of hot distilled water and heat to boiling. Cool to 50°C, add 20.0 mL of 1N HCl, further cool, and add 1.0 g of sodium metabisulfate. Refrigerate in the dark until the solution turns pale yellow (approximately 48 hours). Next, add 5 g of activated charcoal, stir thoroughly, and remove the charcoal by filtration. The clear filtrate is Schiff's reagent, which is storage stable for many months, e.g., at least one year. Furthermore, the resulting magenta color is stronger than that obtained with conventionally prepared Schiff's reagent.

[0039] Galactose oxidase test strips The present disclosure provides test strips or membranes pre-embedded with galactose oxidase. A separate galactose oxidase solution is not required. The galactose oxidase in the test strip or membrane may be encapsulated and may be activated by moisture or contact with water. The amount of galactose oxidase in the test strip or membrane is not limited, so long as it is sufficient to oxidize the marker carbohydrate in the sample.

[0040] In one aspect, the test strips or membranes of the present disclosure can be designed to have different fluid uptakes to accommodate different amounts of marker carbohydrates in different mucus or bodily fluids. For example, a strip or membrane for testing rectal mucus can have different fluid uptakes than a strip or membrane for testing breast secretions. In another aspect, the amount of galactose oxidase in the test strip or membrane can be varied to accommodate different amounts of marker carbohydrates in different mucus or bodily fluids. It can also be varied based on the concentration of the marker carbohydrate in the mucus or body fluid. In another embodiment, the test strip or membrane further comprises a dried culture medium that can activate galactose oxidase upon addition of water onto the test strip or membrane.

[0041] Galactose oxidase test strip device The present disclosure provides a device comprising a test strip or membrane pre-embedded with galactose oxidase and a container containing a Schiff's reagent solution, wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane and can be activated to contact the test strip or membrane after the marker carbohydrate has been oxidized by the galactose oxidase.

[0042] The mechanism for activating the Schiff's reagent solution (contacting it with the test strip or membrane after the marker carbohydrate has been oxidized by galactose oxidase) is not limited. In one embodiment, the mechanism can be a twist valve, a breakable barrier, or the like, where the Schiff's reagent solution contacts the test strip or membrane after opening the twist valve or breaking the barrier. In another embodiment, the mechanism for activating the Schiff's reagent solution is manually transferring the Schiff's reagent solution onto the test strip or membrane. In one embodiment, the device of the present invention includes all components in a single unit to minimize sampling or procedural errors.

[0043] In one embodiment, the galactose oxidase pre-embedded test strip or membrane further contains a dried culture medium, which can activate the galactose oxidase when water is added onto the test strip or membrane. In another embodiment, the pre-embedded galactose oxidase of the test strip or membrane is microencapsulated. In yet another embodiment, the pre-embedded galactose oxidase of the test strip or membrane can be activated by adding a few drops of water onto the test strip or membrane, or by adding mucus or body fluid onto the test strip or membrane.

[0044] In one aspect, the present disclosure relates to the use of the device to screen for cancerous or pre-cancerous conditions, including but not limited to the type of cancerous or pre-cancerous condition, given that marker carbohydrates are present in mucus or bodily fluids of cancerous or pre-cancerous conditions of, for example, the rectum, colon, blood, lymph nodes, stomach, kidney, gallbladder, prostate, testicles, breast, cervix, and ovary.

[0045] In certain embodiments, the galactose oxidase pre-embedded test strip or membrane further contains a pre-embedded Schiff's reagent, in which the pre-embedded galactose oxidase is activated prior to activation of the pre-embedded Schiff's reagent, such that reaction of the sample with galactose oxidase occurs prior to reaction with the Schiff's reagent.

[0046] Embedding and Encapsulation As discussed above, in certain embodiments, galactose oxidase is pre-embedded in the test strip or membrane. In some embodiments, galactose oxidase is directly embedded, absorbed, coated, or impregnated into the membrane or test strip. In certain embodiments, galactose oxidase is encapsulated before being embedded, absorbed, coated, or impregnated into the membrane or test strip.

[0047] In embodiments in which the galactose oxidase is encapsulated or microencapsulated, the encapsulating material may be one or more of the following: It can be made of the above polymers.

[0048] In certain embodiments, the encapsulating material can be prepared according to the methods of Caruso (Phys. Chem. Chem. Phys., 2011,13, 4782), Sukhishvili (Chem. Mater., 2006, 18 (2), 328), US Patent Application Publication No. 20150164805, EP 2213280, or Schwendeman (J Control Release. 2014;196:60). Materials used to prepare encapsulating materials include emulsifiers, materials with different melting points, materials with different hydrophilic / lipophilic balances (HLB), phospholipids, fatty acids, plant sterols, sorbitan esters, beeswax, carnauba wax, paraffin, stearates, shellac, cellulose derivatives, maltodextrin, starch, gum, cellulose, polypyrrole, polycarbonate, cetyltrimethylammonium halides, silanes, diblock copolymers, triblock copolymers, such as poly(ethylene oxide)-block-poly(ethylene oxide) copolymers designated as P123 (PEO20PPO70PEO20) and F127 (PEO106PPO70-PEO106). (propylene oxide)-block-poly(ethylene oxide), alginate, chitosan, xanthan gum, polysaccharides, polysaccharide hydrogels, poly(lysine), poly(acrylic acid), agarose, PEG, poly(hydroxyethyl methacrylate-methyl methacrylate), poly(acrylic acid-co-acrylamide), poly(allylamine hydrochloride), poly(styrenesulfonic acid sodium salt), poly(diallyldimethylammonium chloride), poly(ethyleneimine), N-hydroxysuccinimide-PEG, maleimide-PEG conjugated phospholipids, paraffin, cyclodextrin, carboxymethylated polysaccharides, polycaprolactone, humic substances, Span 60, cholesterol, N-trimethylchitosan chloride, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), poly(N-isopropylacrylamide), poly(N-isopropylmethacrylamide), poly(Nn-propylacrylamide), carboxymethylcellulose, plastic, gold, molecular weight cutoff filter, organic / inorganic hybrid material These include (but are not limited to) materials, metal oxides, plastics, silicas including SBA-15 (PD 50 Å to 89 Å) and MCM-41, ceramics, clays, smectic clays and niosomes.

[0049] In certain embodiments, the encapsulating material has or is later modified to present functional groups that can subsequently react with the membrane or test strip using standard synthetic reactions. For example, in certain embodiments, the encapsulating material may have aminoalkyl, ester, amide, or carbamate functional groups that can react with active groups on the membrane or test strip, resulting in immobilization of the reagent. Numerous standard coupling methods are described, including, but not limited to, March (Advanced Organic Chemistry, 3rd Edition, Wiley, New York, 1985), Odian (The Principles of Polymerization, 2nd Edition, Wiley, New York, 1981), and Bioconjugate Techniques (Hermanson, G.T., Bioconjugate Techniques; Academic Press: San Diego, 1996). Not known in the literature.

[0050] Other methods of encapsulation include, but are not limited to, those disclosed in U.S. Patent Application Publication No. 20160075976 or U.S. Patent No. 6,258,870, each of which is incorporated herein by reference.

[0051] polymer materials Suitable thermoplastic polymers for incorporation as an encapsulating material include polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylenes, Examples include, but are not limited to, succinates, poly(malic acid) polymers, polymaleic anhydrides, poly(methyl vinyl) ethers, poly(amino acids), chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials.

[0052] Examples of biodegradable polymers and oligomers suitable for use in the compositions and methods of the present invention include, but are not limited to, poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); and poly(lactic-co-glycolic acid); poly(caprolactone); poly(malic acid); polyamides; polyanhydrides; polyamino acids; polyorthoesters; polyetheresters; polycyanoacrylates; polyphosphazines; polyphosphoesters; polyesteramides; polydioxanone; polyacetals; polyketals; polycarbonates; polyorthocarbonates; degradable polyurethanes; polyhydroxybutyrates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; chitin; chitosan; oxidized cellulose; and copolymers, terpolymers, blends, combinations, or mixtures of any of the above materials.

[0053] As used herein, "hydrophobic" refers to a polymer that is not substantially soluble in water. As used herein, "hydrophilic" refers to a polymer that may be water-soluble or that has an affinity for absorbing water, but that will draw water into the device, typically not when covalently bonded to a hydrophobic moiety as a copolymer.

[0054] Hydrophilic polymers suitable for use herein can be obtained from a variety of commercial, natural, or synthetic sources known in the art. Suitable hydrophilic polymers include polyanions, including anionic polysaccharides such as alginate; agarose; heparin; polyacrylates; polymethacrylates; ethylene maleic anhydride copolymer (half ester); carboxymethyl amylose; carboxymethyl cellulose; carboxymethyl dextran; carboxymethyl starch; carboxymethyl chitin / chitosan; carboxycellulose; 2,3-dicarboxycellulose; tricarboxycellulose; carboxy gum arabic; carboxy carrageenan; carboxy pectin; carboxy tragacanth gum; carboxy xanthan gum; carboxy guar gum; carboxy starch; pentosan polysaccharides. Examples of hydrophilic polymers include, but are not limited to, cellulose sulfate, curdlan, inositol hexasulfate, β-cyclodextrin sulfate, hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, dextran sulfate, heparin sulfate, carrageenan, polygalacturonate, polyphosphate, polyaldehyde-carbonate, poly-1-hydroxy-1-sulfonate-propene-2, copolystyrene maleate, mesoglycan, sulfopropylated polyvinyl alcohol, cellulose sulfate, protamine sulfate, phosphoguar gum, polyglutamic acid, polyaspartic acid, polyamino acids, and any derivatives or combinations thereof. Those skilled in the art will recognize other hydrophilic polymers that are also within the scope of the present invention.

[0055] Various water-soluble polymers suitable for use herein include, but are not limited to, poly(alkylene glycols), polyethylene glycol ("PEG"); propylene glycol; ethylene glycol / propylene glycol copolymers; carboxymethyl cellulose; dextran; polyvinyl alcohol ("PVOH"); polyvinylpyrrolidone; poly(alkyleneamines); poly(alkylene oxides); poly-1,3-dioxolanes; poly-1,3,6-trioxanes; ethylene / maleic anhydride copolymers; polyamino acids; poly(n-vinylpyrrolidone); polypropylene oxide / ethylene oxide copolymers; polyoxyethylated polyols; polyvinyl alcohol succinate; glycerin; ethylene oxide; propylene oxide; poloxamers; alkoxylated copolymers; water-soluble polyanions; and any derivatives or combinations thereof. Additionally, the water-soluble polymer may be of any suitable molecular weight and may be branched or unbranched.

[0056] Depending on the desired softness and flexibility of the encapsulating material, the rate and extent of reagent release, degradation rate, etc., the amount and type of polymer can be varied to achieve the desired results.

[0057] Encapsulation Shell and Impregnation Layer In certain embodiments, the polymer becomes porous over time under certain conditions, thereby forming an encapsulating shell that can control release. The pores can be formed by swelling of the polymer shell or by dissolution or degradation of the shell.

[0058] The mass, volume and thickness of the polymer in each encapsulating shell / sphere can also be varied to tailor the release rate of the incorporated reagent.

[0059] The use of the term shell / sphere, as used herein, is not limiting with respect to the shape of the encapsulating material. The shape of the material is generally spherical, although conical shells, tubular shells, oval shells, cylindrical rods, and the like can be made and used. In certain embodiments, the material may be amorphous or irregular. In certain other embodiments, the encapsulating material may be coated or adhered to the surface of a scaffolding or chromatographic material. In such embodiments, the encapsulating material may take the shape of the material to which it adheres. In certain embodiments where the chromatographic or scaffolding material is porous, the encapsulating material may or may not penetrate the pores of the underlying material.

[0060] In certain other embodiments, a polymer can be impregnated with the reagent and coated onto the surface of a membrane or test strip. In such embodiments, the reagent is blended or mixed with the polymer such that the reagent is embedded, encapsulated, or impregnated into the polymer matrix. In such embodiments, the encapsulating material does not form a discreet encapsulating shell, but instead, the encapsulating material containing the reagent is coated as a layer onto the membrane or test strip and may optionally be covalently or ionically bonded thereto.

[0061] In certain embodiments, the encapsulating material may be wax, hydrogel, silicone rubber, or trehalose glass. While the use of hydrogel, silicone rubber, and trehalose glass are particularly suited to impregnating the polymeric material with reagents, any suitable polymer may be used in such embodiments.

[0062] In yet other embodiments, the reagents may be loaded into the pores of the porous membrane or test strip material, in which case the porous material may be coated with one or more polymeric encapsulating materials as described herein after the reagents have been loaded into the pores of the porous material.

[0063] Induction of release The encapsulated galactose oxidase reagent can be released from the encapsulating material by a variety of means known to those skilled in the art. In certain embodiments of the present invention, such release can be induced by contacting the encapsulating material with a pore-forming agent. In other embodiments of the present invention, release can be induced by a physical or chemical change. For example, but not limited to, release can be induced by a change in temperature, pH, ionic charge, counterion charge, or counterion atom. Similarly, release can be induced by contacting the encapsulating material with an organic solvent, aqueous solvent, aliphatic solvent, aromatic solvent, oxygenated solvent, or the like. Alternatively, release can be induced by contact with solvents including, but not limited to, halogenated solvents, or water.

[0064] Generally, the release rate of the reagents will be determined by one of skill in the art based on the membrane or test strip used. In certain embodiments, the desired release rate is immediate, while in other embodiments, the release rate is controlled so that the reagents are released over a period of time. In certain embodiments, the reagents are released during the course of the test / workflow so that about 100% of the reagents are released by about 100% of the sample being introduced. In other embodiments, the reagents are released during the course of the test / workflow so that about 100% of the reagents are released by about 90% of the sample being introduced, about 80% of the sample being introduced, about 75% of the sample being introduced, about 50% of the sample being introduced, or about 25% of the sample being introduced.

[0065] Pore-forming agents Other additives can be advantageously used to further control the desired release rate of the reagent for a particular test / workflow protocol. For example, if the thermoplastic polymer liquid composition is too impermeable to water, a pore-forming agent can be added to generate additional pores in the matrix. Any suitable water-soluble material can be used as the pore-forming agent. These agents can be either soluble in the liquid composition or simply dispersed therein. These agents can dissolve, diffuse, or disperse from both the solidified polymer matrix and the formed polymer system, resulting in the generation of pores and microporous channels within the matrix and system. The amount of pore-forming agent in the composition (and, if appropriate, the size of dispersed particles of such pore-forming agent) directly affects the size and number of pores within the polymer system.

[0066] Other factors can also affect the size and / or diameter of the pores formed within the polymer system. For example, the amount of organic solvent and the rate at which the polymer system solidifies can both affect the porosity of the polymer system. While generally microporous matrices without a decomposed core and skin can be produced according to the present invention, typically, without the use of an additional pore-forming agent, polymer systems formed from the liquid composition will consist of a surface skin and an inner core. The surface skin is typically less porous, or even relatively non-porous, compared to the inner core. The inner core may have pores with diameters ranging from about 10 μm to 1000 μm. The use of an additional pore-forming agent results in the core and skin having substantially uniform pore sizes, both of which have pores in the range of 10 μm to 1000 μm.

[0067] The concentration of pore-forming agent relative to the thermoplastic polymer in the composition will vary depending on the degree of pore formation desired. Generally, this concentration will range from about 0.01 g to 1 g of pore-forming agent per gram of polymer. If the agent is soluble in the liquid composition, the mixing or dispersion of the agent in the liquid composition and its coagulation when the thermoplastic polymer solidifies will determine the size of the pores obtained when the agent dissolves from the polymer matrix.

[0068] Pore-forming agents include any pharmaceutically acceptable organic or inorganic material that is substantially miscible with water and body fluids and dissipates from the formed matrix into aqueous media or body fluids, or into water-immiscible materials that rapidly degrade into water-soluble materials. The pore-forming agent may be soluble or insoluble in the polymer liquid composition of the present invention. It is further preferred that the pore-forming agent in the liquid composition of the present invention is miscible or dispersible in organic solvents to form a uniform mixture. Suitable pore-forming agents include, for example, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, and polymers such as hydroxypropyl cellulose, carboxymethyl cellulose, polyethylene glycol, and polyvinylpyrrolidone. The molecular weight and ratio of the pore-forming agent incorporated into the polymer system can be varied. By varying the size and range of the pores, it is possible to vary the size and range of the pores widely.

[0069] Other excipient materials, such as sucrose, dextrose, sodium chloride, sorbitol, lactose, polyethylene glycol, mannitol, fructose, polyvinylpyrrolidone, or suitable combinations thereof, can be added to the device to vary the porosity. Additionally, the active agent can be dispersed in oils (e.g., sesame oil, corn oil, vegetable oil), or mixtures thereof with phospholipids (e.g., lecithin), or medium-chain triglycerides (e.g., Miglyol 812) to obtain an oily suspension.

[0070] Test Method The present disclosure provides a method for rapidly detecting the expression of marker carbohydrates in a subject using a test strip or membrane pre-embedded with galactose oxidase and a Schiff's reagent solution.

[0071] The present disclosure also provides a method for rapidly testing for cancerous or pre-cancerous conditions in humans using a test strip or membrane pre-embedded with galactose oxidase and a Schiff's reagent solution.

[0072] In one aspect, the method comprises the steps of applying mucus or body fluid to a test strip or membrane pre-embedded with galactose oxidase, oxidizing the marker carbohydrates in the mucus or body fluid containing the marker carbohydrates by reacting them with the galactose oxidase, and activating a reservoir containing a Schiff's reagent solution adjacent to the test strip or membrane to contact the test strip or membrane, wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane.

[0073] In some embodiments, the method includes one or more steps selected from applying water onto the test strip or membrane before applying the mucus or body fluid to the test strip or membrane, removing the mucus or body fluid by washing before activating the container containing the Schiff's reagent solution, rinsing the test strip or membrane with tap water after contacting the Schiff's reagent solution with the test strip or membrane, drying the test strip or membrane, and / or evaluating the color of the test strip or membrane.

[0074] In one embodiment, the time for oxidizing the marker carbohydrate in mucus or body fluid by reacting it with galactose oxidase is not particularly limited. For example, the oxidation time can be 3 to 30 minutes, 5 to 20 minutes, 7 to 15 minutes, or 8 to 12 minutes. In another embodiment, the time for contacting the Schiff's reagent solution with the test strip or membrane is not particularly limited. For example, the contact time can be 0.2 to 10 minutes, 0.5 to 5 minutes, 0.8 to 3 minutes, or 1 to 2 minutes.

[0075] In another embodiment, the mechanism for activating the Schiff's reagent solution (contacting it with the test strip or membrane after the marker carbohydrate has been oxidized by galactose oxidase) is not limited. In one embodiment, the mechanism can be a twist valve, a breakable barrier, or the like, where the Schiff's reagent solution contacts the test strip or membrane after opening the twist valve or breaking the barrier. The material of the breakable barrier is not limited, and the breakable barrier can be made of plastic, glass, or any material suitable for liquid containers.

[0076] Test kit The present disclosure provides a screening kit for rapidly detecting the expression of marker carbohydrates in a subject using a test strip or membrane pre-embedded with galactose oxidase and a Schiff's reagent solution.

[0077] The present disclosure further provides a screening kit for rapidly testing for a cancerous or precancerous condition in humans, the screening kit comprising a test strip or membrane pre-embedded with galactose oxidase and a container containing a solution of Schiff's reagent.

[0078] In one embodiment, the galactose oxidase pre-embedded test strip or membrane further contains a dried culture medium that can activate the galactose oxidase upon addition of water onto the test strip or membrane, hi another embodiment, the pre-embedded galactose oxidase of the test strip or membrane is microencapsulated.

[0079] In one aspect, the present disclosure relates to the use of the test kit for screening for cancerous or precancerous conditions, without limitation of the type of cancerous or precancerous condition, considering marker carbohydrates present in mucus or body fluids of cancerous or precancerous conditions of, for example, the rectum, colon, blood, lymph nodes, stomach, kidney, gallbladder, prostate, testicles, breast, cervix, and ovary. [Example]

[0080] Example 1 - A mucosal scraping sample is collected from a subject. The scraping sample is mixed with distilled water or reverse osmosis water and applied to a test strip pre-embedded with galactose oxidase at a concentration of 100 U / mL. The sample is left on the test strip for 10 minutes, after which the test strip is rinsed with additional distilled water. In a separate container, Schiff's reagent is activated in solution, and then the solution is added to the test strip. The sample is left in contact with Schiff's reagent for 1 minute, after which the test strip is rinsed with water and dried in open air or in an oven. Figure 2 illustrates the test sample processing. A color change (from white or colorless to magenta) indicates the presence of the carbohydrate marker.

[0081] Other documents cited herein are incorporated herein in their entirety by reference to the extent such disclosure does not contradict this disclosure and for all jurisdictions where such incorporation is permitted.

[0082] Where numerical lower limits and numerical upper limits are recited herein, ranges from any lower limit to any upper limit are contemplated. While exemplary embodiments of the present disclosure have been described in detail, it is understood that various other modifications will be apparent to those skilled in the art and may be readily made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the scope of the claims appended hereto be limited to the examples and illustrations set forth herein, but rather that the claims be construed to encompass all patentably novel features present in the present disclosure, including all features that would be treated as equivalents by one skilled in the art to which the present disclosure pertains.

[0083] The present disclosure has been described above with reference to numerous embodiments and examples. Many modifications will occur to those skilled in the art in light of the above detailed description. All such obvious modifications are intended to be included within the full scope of the appended claims.

Claims

1. 1. A screening device for rapidly testing human mucosal carbohydrate expression, comprising: a test strip or membrane pre-embedded with galactose oxidase; a container containing a Schiff's reagent solution; wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane, and after the marker carbohydrate is oxidized by galactose oxidase, the Schiff's reagent solution can be activated and brought into contact with the test strip or membrane.

2. 1. A screening device for rapidly testing for a cancerous or precancerous condition in a human, comprising: a test strip or membrane pre-embedded with galactose oxidase; a container containing a Schiff's reagent solution; wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane, and after the marker carbohydrate is oxidized by galactose oxidase, the Schiff's reagent solution can be activated and brought into contact with the test strip or membrane.

3. The screening device according to claim 1 or 2, wherein the galactose oxidase is microencapsulated.

4. 3. The screening device of claim 1, wherein the test strip or membrane further comprises a dried culture medium, the dried culture medium being capable of activating the galactose oxidase upon addition of water onto the test strip or membrane.

5. 3. The screening device of claim 1 or 2, wherein the Schiff's reagent solution is activated by opening a twist valve or by disrupting a barrier between the Schiff's reagent solution container and the test strip or membrane.

6. 3. The screening device of claim 1, wherein the Schiff's reagent solution is a storage-stable Schiff's reagent solution.

7. 1. A screening method for rapidly testing human mucosal carbohydrate expression, comprising: applying the mucus or body fluid to a test strip or membrane pre-embedded with galactose oxidase; oxidizing marker carbohydrates in the mucus or body fluid by reacting them with galactose oxidase; activating a reservoir containing a Schiff's reagent solution adjacent to the test strip or membrane to contact the test strip or membrane; wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane.

8. 1. A screening method for rapidly testing for a cancerous or precancerous condition in a human, comprising: applying the mucus or body fluid to a test strip or membrane pre-embedded with galactose oxidase; oxidizing marker carbohydrates in the mucus or body fluid by reacting them with galactose oxidase; activating a reservoir containing a Schiff's reagent solution adjacent to the test strip or membrane to contact the test strip or membrane; the mucus or body fluid contains a marker carbohydrate, and the Schiff's reagent solution is initially A screening method that does not involve contact with test strips or membranes.

9. 9. The screening method of claim 7 or 8, further comprising applying water onto the test strip or membrane before applying the mucus or body fluid to the test strip or membrane.

10. The screening method according to claim 7 or 8, wherein the galactose oxidase is microencapsulated.

11. 9. The screening method of claim 7 or 8, wherein the test strip or membrane further contains a dried culture medium, which can activate the galactose oxidase when water is added onto the test strip or membrane.

12. 12. The screening method of claim 11, further comprising applying water onto the test strip or membrane before applying the mucus or bodily fluid to the test strip or membrane.

13. 9. The screening method of claim 7 or 8, wherein the container containing the Schiff's reagent solution adjacent to the test strip or membrane is activated by opening a twist valve or by disrupting a barrier between the container of the Schiff's reagent solution and the test strip or membrane.

14. 9. The screening method according to claim 7, wherein the marker carbohydrate in the mucus or body fluid is oxidized by reacting it with galactose oxidase for 5 to 20 minutes.

15. 9. The screening method according to claim 7 or 8, further comprising removing the mucus or body fluid by washing before activating the container containing the Schiff's reagent solution.

16. 9. The screening method of claim 7 or 8, further comprising contacting the Schiff's reagent solution with the test strip or membrane for 0.5 to 5 minutes.

17. 17. The screening method of claim 16, further comprising, after contacting the Schiff's reagent solution with the test strip or membrane for 0.5 to 5 minutes, rinsing the test strip or membrane with tap water, drying the test strip or membrane, and evaluating the color of the test strip or membrane.

18. A screening kit for rapidly testing human mucosal carbohydrate expression, comprising: a test strip or membrane pre-embedded with galactose oxidase; a container containing a Schiff's reagent solution; A screening kit comprising:

19. 1. A screening kit for rapidly testing for a cancerous or precancerous condition in a human, comprising: a test strip or membrane pre-embedded with galactose oxidase; a container containing a Schiff's reagent solution; A screening kit comprising:

20. 20. The screening kit according to claim 18 or 19, wherein the galactose oxidase is microencapsulated.

21. 20. The screening kit of claim 18 or 19, wherein the test strip or membrane further comprises a dried culture medium, which can activate the galactose oxidase upon addition of water onto the test strip or membrane.

22. Use of the screening device of claim 2 for screening for a cancerous or precancerous condition.

23. 23. Use of a screening device according to claim 22, wherein the cancerous or precancerous condition is a cancerous or precancerous condition of the rectum, colon, blood, lymph nodes, stomach, kidney, gallbladder, prostate, testicle, breast, cervix or ovary.

24. Use of the screening kit of claim 19 for screening for a cancerous or precancerous condition.

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