TW201109655A - Assay device and methods - Google Patents

Abstract

The present invention relates to the use of amphipathic polymers to enhance lateral flow and reagent mixing on assay devices. More specifically, the invention relates to use of an amphipathic polymer in assay methods including a device for determining the concentration of lipids in blood serum or plasma.

Description

201109655 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to the use of amphiphilic polymers for enhancing lateral articulation and reagent mixing on assay devices. More specifically, the present invention relates to the use of amphiphilic polymers in assay methods comprising means for determining the lipid concentration in serum or plasma. [Prior Art] Lateral flow measuring devices and methods are well known to those skilled in the art. Previously, such devices have been developed to test samples that are readily available in large quantities. However, when the test sample is blood or blood components, it is not always possible to collect a large number of samples, especially at the point of care (eg, doctor's office). Typically, such devices comprise a lateral flow matrix, such as a nitrocellulose membrane and the like. A sample applied to the substrate flows along the substrate and one or more analytes in the sample are ruminant with one or more reagents in the lateral flow matrix. Typically, at least one of the agents is immobilized within the matrix, such that any reaction with the analyte can be visually detected, for example. The 4 撼 S, sample transfer and sample-to-membrane diffusion differences result in extremely uncontrolled and non-uniform flow before reaching the test area. This is because these devices rely solely on the capillary function of the fluid. This dependence on capillary action may have an adverse effect on device accuracy, as the analytes and/or & The use of capillary action alone also means that the measurement is slower because fluid wicking is not reliable. These measurements are also not applicable to small fluid samples', e.g., in nucleic acid detection, where the membrane may have dried or not sufficiently fluid to travel the length of the test device prior to completion of the assay. I45179.doc 201109655 Therefore, there is a need for a simple and efficient technique for improving lateral flow measurements to allow for rapid testing while enabling low volume testing. One area in which a lateral grasping 4-point device may be useful is in the field of cholesterol and blood lipid testing. Those skilled in the art are aware that ‘, A ^ Λ no, knows the concentration of various lipoproteins in the blood and the risk of atherosclerosis in the individual. Atherosclerosis is a disease that affects the arteries of the arteries and is often referred to as arterial "hardening". (4) The formation of multiple plaques on the wall of the hard MUM (to a large extent due to the aggregation of giant blood cells, 2 丨 杲 杲), and low-density lipoprotein will accelerate atherosclerosis. When high-density lipoprotein (progenitor) does not adequately remove fat and cholesterol from giant cells, a chronic inflammatory response occurs in the arterial wall. Most circulating cholesterol in plasma is found in three major lipoproteins. Cholesterol and cholesterol § is intended to be water-insoluble (iv) f, I therefore m is carried by these lipoproteins and ultimately utilized by the body cells. Each of these scorpion protein species carries a different amount of cholesterol. Therefore, the total serum cholesterol is a combined average of the amounts of various lipoproteins that contribute to serum total lipoprotein concentrations. Various types of lipoproteins differ in atherosclerosis. High-density lipoproteins or ancestors are often considered "good cholesterol", that is, their resistance to atherosclerosis. Phase & 'Low density lipoprotein or ldl is commonly referred to as "bad cholesterol" because it is known to be highly atherogenic. Another type of lipoprotein, very low density lipoprotein or VLDL, is considered to be slightly atherogenic. Considering the inverse relationship between HDL cholesterol and atherosclerosis and, for example, heart attack 145179.doc 201109655 risk has been extensively studied for hdl content in liquids. Therefore, if the level of HDL cholesterol is determined to be low, the individual may have an increased risk of developing atherosclerosis. Therefore, the risk can be assessed by measuring HDL cholesterol. From the results of these measurements, the following formula can be used to calculate the approximate amount of LDL cholesterol: LDL cholesterol = total cholesterol - 1 / 5 total cholesterol cholesterol Four methods are commonly used to determine the cholesterol content of each cholesterol fraction. The four methods include (1) ultracentrifugation, (2) stepwise precipitation, (3) calculation using the Friedewald formula, and (4) electrophoretic separation and precipitation. Each of these methods has a number of disadvantages. For example, ultracentrifugation requires special laboratory equipment and can take several days to complete. Both stepwise precipitation and electrophoretic separation take time and require special equipment. (d) The dewaid formula is not accurate because it assesses the concentration of LDL cholesterol by subtracting cholesterol associated with other lipoproteins. Therefore, the 兮8 formula provides an indirect assessment based on three independent lipid analyses, and there is a potential source of error for each lipid analysis. Due to these shortcomings, the Helmets method obtains cholesterol measurements within hours or even days and cannot be counted within the small-test or by the doctor in the clinic. Therefore, there is a need for an apparatus and method for performing cholesterol testing that is early and inexpensive. It is also necessary to directly measure the various lipoproteins of jilu θa base.

Determination independent of indirect evaluation β X When a solid hydrophobic molecule is added to a liquid, a suspension or a suspension, the hydrophobicity is "sticky" together instead of , B p ^ is not / combined, It immediately forms a precipitate and does not enter the aqueous phase. Even with vigorous agitation, the number of direct encounters between the hydrophobic molecule and the solution application 145I79.doc 201109655 is very small. These interactions are also thermodynamically unfavorable. Prior art methods involve dissolving the hydrophobic molecule in a suitable water miscible organic solvent prior to mixing with the aqueous solution or suspension. Upon entering the aqueous solution, the hydrophobic molecules are monodisperse and thus increase the likelihood of interaction with molecules already in solution. However, these methods have the disadvantage that organic solvents are generally toxic or may interfere with enzymatic or fluorescent measurements. These methods are generally not suitable for use at a point of care (for example, at a doctor's office or clinic). SUMMARY OF THE INVENTION Applicants have obtained the surprising discovery that the use of an amphiphilic polymer in a hydrazine system results in a substantial increase in both measurement efficiency and/or speed. These methods of the Applicant are also applicable to the rapid incorporation of hydrophobic reagents and compounds (e.g., luminophores) with analytes (e.g., lipoproteins) in aqueous solutions/suspensions without the direct application of organic solvents. The method is capable of easily measuring the blood, food, and the like of the lipoprotein by the amount of fluorescence. Using these methods, it is also possible to develop a rapid and inexpensive point-of-care cholesterol measuring system. In a first aspect of the invention, there is provided an assay device for detecting the presence or amount of an analyte present in an aqueous sample, the device comprising: a flow path along which at least one aqueous sample can travel, characterized in that The at least one flow path comprises at least one amphiphilic polymer, wherein the fluid passing along the flow path in use is greater than the polymer having both hydrophilicity and hydrophobicity by the expectant/parental polymer system which is solely capillary-only . This & substance can also be referred to as an amphiphilic compound or a nonionic hydrophilic 145179.doc 201109655 polymer. In a particular embodiment, the amphiphilic polymer is a material that is soluble in water and a wide range of organic solvents. The present invention relates to the use of amphiphilic polymers (e.g., polyethylene glycol) for promoting and/or controlling lateral fluid flow. The present invention can also be utilized to increase the interaction between an aqueous sample and at least one reagent, preferably a hydrophobic luminophore, and a lipoprotein. Preferably, the flow path is coated with at least one amphiphilic polymer. Surprisingly, it has been found that an amphiphilic polymer can be used to coat the surface of, for example, a plastic or glass to enhance fluid flow and thereby move the aqueous solution and/or to mix it with, for example, a dry, "dry" component. The components are combined within the amphiphilic polymer coating or as a layer above or below the coating. The use of amphiphilic polymers also has the advantage that lateral fluid flow is improved, for example, superior to the conventional "wicking" of porous materials disclosed in, for example, U.S. Patent No. 6,485,982. Prior art wicking methods rely on the use of a support carrier, such as paper or film, which draws liquid through the support carrier by capillary action. The use of an amphiphilic polymer removes the need for a support carrier that relies solely on capillary action' and has the surprising effect that the liquid travels along, for example, microtubules or surfaces rather than by a single capillary-only effect. The distance traveled in the material is farther or faster. As used herein, the term "lateral flow" refers to the movement of a fluid in a particular direction or along a particular path (e.g., laterally) on a fluid flow surface. Preferably, the 'grass reading' flows or flows a greater distance only by the speed at which the capillary is observed through a particular material. It should be noted that the term "lateral flow" is intended to be descriptive and not limiting, as the device may be constructed in other manners with the same effect as the device 145179.doc 201109655', for example, the same as the present invention can be easily utilized. The principle envisions radial flow or vertical flow 'this is not surprising to the spirit of the invention. In particular embodiments, as the fluid flows or advances, it interacts with the various reagents simultaneously, and the analytes contained therein can react with the various reagents. The apparatus includes at least one flow path along which the aqueous sample can travel, and wherein the at least one flow path comprises at least one amphiphilic polymer wherein the fluid passing along the flow path in use is greater than by capillary action alone Expected. The device does not require a porous membrane to achieve lateral fluid flow as compared to the prior art, and instead the fluid can travel along a flow path comprising at least 2 amphiphilic polymers. Surprisingly, it has been found that the flow of fluid along a flow path comprising an amphiphilic polymer is faster and/or travels a greater distance than a flow path comprising a porous material (e.g., a membrane). In a particular embodiment, the flow path contains at least one amphiphilic polymer. Amphiphilic polymers can therefore be used to coat surfaces such as tubes, burrs, channels, wells, membranes or the like. It will be appreciated that amphiphilic polymers can also be used as a coating for membranes in "standard" lateral flow assays to accelerate fluid flow and/or provide more control over fluid flow. In other embodiments, the flow path is formed from at least one amphiphilic polymer. The hydrophobic or amphiphilic polymer can be printed and/or sprayed onto the surface by a printing process (eg, by inkjet printing or bubble-jet printing), smearing, spraying, or other application method ( For example, on a flat surface, 145179.doc 201109655, for example, forms a "dead" and/or layer. The amphiphilic polymer can be in the form of a film. In other embodiments, the film can be ground into particulate material or can form, for example, particles, beads, pellets, microspheres, or nanospheres or microspheres. The amphiphilic polymer itself may be in the form of particles, beads, beads, microspheres, nanospheres or microspheres. In still other embodiments, the amphiphilic polymer can be partially or partially printed as one or more nanoliter, picoliter or femtoliter droplet arrays to form one or more nanoliters, picoliters or femto Rise the array of droplets. Preferably, the amphiphilic polymer comprises at least one probe, reporter molecule or reagent. Preferably, the reporter molecule is a hydrophobic compound or reagent (for example, luminescence is obtained by combining a hydrophobic compound or a reagent with a polymer), and the hydrophobic compound or reagent can be first dissolved in a solvent miscible with the amphiphilic polymer ( For example, in an organic solvent, the amphiphilic polymer is also dissolved in a solvent such as water or a volatile solvent such as dimethylformamide or chloroform, but those skilled in the art will be aware of other suitable solvents. The hydrophobic compound or agent is combined with the amphiphilic polymer' and then preferably dried to form a film. Surprisingly, it has been found that the hydrophobic compound or agent does not phase separate from the polymer after drying. =:=: Try a group of wells, membranes or, for example, amphiphilic polymers, so that at least one hydrophobic luminophore can be used to coat the surface, such as the inner side of tubes, capillaries, channels, 145l79.doc 201109655. In still other embodiments, the combination is printed onto the surface by a printing process (eg, by inkjet printing or bubble jet printing), smearing, spraying, or other application method, for example Forming "orbital gamma probes, reporter molecules or reagents may be adjacent to at least one flow enthalpy. Alternatively, probes, reporter molecules or reagents may be layered directly above or below at least one flow path. Because & 'reagents may be flowing In the path itself (for example, in combination with an amphiphilic polymer), the constituent vertical layers may be placed above, below or near the amphiphilic polymer or printed as "dots". It will be appreciated that the method of the invention is also applicable to the removal of hydrophobic light. Other reagents other than the group, such as enzymes, blocking reagents, chemicals, and the like. More preferably, the amphiphilic polymer has a molecular weight of about 1000 Da to 2 〇〇〇〇 Da, and more preferably about 1000 Da to 6000 Da. Moreover, polyethylene glycol (PEG) of about 1000 Da to 3 〇〇〇 Da is particularly preferred. PEGs including PEG2000, PEG6000, PEG12000 and PEG20000 〇 polyethylene glycol are also known as polyethylene oxide (pE〇). Or polyoxyethylene (PQE), which is a polymer of epoxide or a polymer PEG having a molecular weight ranging from 300 g/mol to 10,000,000 g/mol. peg has the following general structure: H0 -(CH2-CH2-0-)nH. Usually included in PEG The number in the middle indicates the average molecular weight. For example, the average molecular weight of PEG of 'n=80 is about 35 〇〇 Daltons, and is called PEG 3500. Usually 'PEG includes molecules with a series of molecular weights, albeit with different molecular weights. PEG is used in various applications due to different physical properties (such as viscosity), but its chemical properties are almost the same. The starting agent used in the polymerization process can also get different forms of PEG. For example, the monofunctional methyl ether PEG (methoxy poly(ethylene glycol)), abbreviated as mPEG. It is also possible to obtain PEG-chained PEGs having different geometries having from 3 to 10 PEG chains emanating from the central core group. Star PEG has from 10 to 100 卩 链 链 发 》 》 梳 梳 PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG. It is also possible to covalently couple PEG to other molecules during a process known as poly 6 diolation, for example, which may be advantageous when utilizing the fluid flow characteristics of PEG for reagent mixing. Other amphiphilic polymers may include amphiphilic polypeptides, i.e., polymorphs having a primary structure, whereby the polypeptide has a hydrophilic surface and a hydrophobic surface. The design of the amphiphilic structure (e.g., an alpha-helical polypeptide) is well known. It is known to the skilled person. For example, Grell et al., (2001) J Pept Sci 7(3): 146 5 1 Chen, (2002) J Pept Res 59(1): 18-33; iwata et al.

Boil Chem 269(7): 4928-33 ; Cornut et al. (1994) FEBS

Lett 340(1): 29-33; Negrete et al., (I%8) Protein Sci 7(6): 1368-79. Other amphiphilic or nonionic polymers include polyvinyl alcohol (PVA) (Sigma Aldrich: 360627-25G), carboxymethyl cellulose (Sigma: C-5678); 0,0,··bis (2-amino group) Ethyl) PEG 2000 (polyoxyethylene bis (amine)) (Aldrich Chemistry: 14501) and PEG methyl 5,000 (Aldrich Chemistry: 81323-250G) and other ion plastic polymers. Preferably, the apparatus further comprises at least one application zone for applying an aqueous sample. The area on the zone system device where the sample is applied is applied. Preferably, the presence of 145179.doc 11 201109655 single application zone' from which multiple (but at least one, two, three or more) sample fractions can be obtained for performing the assay of the method of the invention. Or ' separate or separate sample dispensing has a separate application area. It will be appreciated that in some embodiments the device can be designed to introduce the sample directly into the test zone, thereby avoiding the need for an application zone. As used herein, the term "aqueous sample" means any liquid sample, preferably a liquid sample from which the analyte can be detected. Non-limiting examples of such samples include whole blood, serum or plasma samples, urine, cerebrospinal fluid (CSF), lymph, serous exudate or other biological fluids, animal tissue homogenate, deproteinized tissue Homogenating 'milk, raw eggs, fermentation broth, animal feed and marine feed. It will be appreciated that the sample need only be in at least a substantial portion of the assay in a liquid or aqueous form. Thus, it is contemplated by those skilled in the art that the assay can also be carried out at temperatures above the melting point of a sample (e.g., wax or lipid) which is typically solid at room temperature, for example. Preferably, the apparatus further comprises at least one test area for determining the result and/or progress of the reaction between the one of the aqueous sample and the probe, the reporter or the reagent. The assay region is capable of determining the analyte and the probe. , the result of the reaction between the reporter or reagent and/or the area of the process. The test area can be in contact with the excitation member. Preferably, at least one flow path is in fluid communication with at least one of the application zones and the at least one of the test zones. Preferably, the device comprises a plurality of flow paths and a plurality of test areas. Preferably, the flow path connects the application zone to at least one of the test zones by, for example, fluid communication. H5179.doc •12· 201109655 The flow path can be a channel, a groove, a capillary, a track or a path, and the like. Preferably, the flow path comprises an amphiphilic polymer. The amphiphilic polymer may be in the form of a coating or film on the surface of the flow path, or may be in the form of a powder, a small particle 'microparticle, a nanoparticle, a microparticle or a filler in the flow path cavity. When the amphiphilic polymer is a filler in the cavity, it may completely fill the cavity or may be partially filled with, for example, a slit. Alternatively, the amphiphilic polymer can be a flow path provided, for example, in the form of a track or path (e.g., printed on the lateral flow of the fluid along the surface on which it occurs). The flow path comprising the amphiphilic polymer may further comprise other reagents required for the assay, e.g., at least one hydrophobic luminophore and/or at least one enzyme is different; the il path may comprise different reagents. The flow path may also contain different reagents along its length', for example, allowing for the sequential addition of reagents throughout the lateral flow of the aqueous sample. Alternatively, the flow path to the test zone comprises a separate amphiphilic polymer comprising a combination of amphiphilic polymer and other reagents. The reagents may be in the flow path itself (e.g., combined with an amphiphilic polymer), or may be layered above, below, or near the amphiphilic polymer. Preferably, the results and/or progress of the reaction are determined by means of optical measurements. Alternatively, the results and/or progress of the reaction are determined by visual inspection. More preferably, the results and/or progress of the reaction are determined by means of fluorescence. The test area is preferably arranged to be in optical contact with the excitation member. The test area should be arranged to detect different aspects of the same measurement by self-measurement by means of the detection means or different measurements may have separate test areas. An "excitation member" is a member operable to excite a sample to fluoresce at least one sample component, typically a probe or reporter molecule. 145179.doc •13· 201109655 The term “contact” as used above is not intended to imply or limit to physical contact. In this context, the term can be simply understood as physically moving the test area to the beam path or The test area is illuminated/excited by the beam. The sensing member is operable to detect a component of the fluorescent light emitted by the sample. In a particular embodiment, the measurements can be performed simply by visual inspection. In this case, the excitation member and the detection member may not be required. In a particular embodiment, the device comprises at least two separate components. More preferably, the at least two separate components are readers and test cartridges. Thus, the device can include two or more separate components, such as a reader and a test cartridge adapted to be placed in functional communication with the reader. Preferably, the test can be inserted into the reader, placed on the reader or attached to the reader. The reader can include a docking station member that can be inserted, placed, or attached to the test station. The station member can be a slot. Preferably, the test g is removable from the reader. The test can take a variety of forms, for example, cards, wafers, and granules, and can be constructed from materials well known to those skilled in the art: 丄 丄 丄 cardboard, glass, enamel, plastic, and the like. Preferably, material L contains either a hydrophobic material or a hydrophobic region. : The good test 'test 匣 contains at least one flow path. Preferably, the disposable type is tested and replaced with a new one that contains a new lookup agent. Preferably, the reader is a reusable component. In the embodiment: the skirt is used for detecting the presence or quantity of the aqueous sample knife, comprising: (1) at least one application 145179 suitable for applying the aqueous sample to the device. Doc •14- 201109655 domain; (9) at least one probe, reporter molecule or reagent in which the probe, reporter molecule or reagent can be reacted with an analyte present in an aqueous sample; (iv) at least- Test areas in which the results and/or progress of the reaction between the analyte and at least one of the probes, reporter molecules or reagents can be determined; (1V) at least one and at least one application zone and at least one test zone A flow path in fluid communication, characterized in that the at least one flow path # comprises at least one amphiphilic polymerization 'and wherein the fluid passing along the at least one flow path when in use is greater than expected by capillary action alone. In a particular embodiment, the apparatus includes a single application zone and a plurality of (e.g., four) flow paths leading to a plurality (e.g., at least three) of test zones. Preferably, when the skirt comprises at least three test zones and at least three flow paths, preferably the flow control is in fluid communication with the application zone and the first test zone. The second flow path is in fluid communication with the application zone and the second test zone. And the third flow path is in fluid communication with the application region and the third test region. In one embodiment, the first flow path and/or test region comprises two: a polymer and a first hydrophobic luminophore and optionally a group of at least one enzyme. The first flow path and/or the test region comprises an amphiphilic polymer. In combination with the second, X) germinating group and optionally at least one enzyme. The third flow path and/or test region comprises an amphiphilic polymer and a first, second or third hydrophobic 145179.doc -15-201109655 luminescent luminophore and, as the case may be, at least a combination of A sample of the application zone (in aqueous form) is flowed into the respective test zone along at least one flow path by lateral flow (achieved by the amphiphilic polymer). When the sample flows along at least one of the flow paths, it is provided with hydrophobic luminescence: and/or other reagents and mixed with the hydrophobic I photo group and/or other reagents. Or 'providing a hydrophobic luminescent group and/or other reagents only when the sample reaches the test area. Preferably, the sample is moved by lateral flow and does not require an external force (e.g., 'pump) to move the aqueous sample. The measurement can be performed after the aqueous sample reaches the test area. When assaying lipid distribution assays, the probe, reporter molecule or reagent may be selected from the group consisting of Amplex Red, K37, Nile Red, Cholesterol Esterase, Cholesterol Oxidase or Horseradish Peroxidase.

Amplex Red (10-ethenyl-3,7-dihydroxyphenazine) is especially available from Invitrogen (catalog numbers A12222 and a22177), which is 1:1 stoichiometric with hydrogen peroxide (H2〇2). The specific reaction produces a highly fluorescent resorufin. K37 (4-didecylamino-4, bis-methylmethyl- sulphate-sub-phenylidene-acetophenone) is disclosed by the inventors in its prior international patent application PCT/GB2005/004757. The Nile red lipophilic dye, also known as Nile Blue Oxazinone, can be purchased from 11^"1*(^11(catalog number)^1142) or by boiling boiled Nile Blue The solution is produced. Nile red dyes intracellular lipid droplets in red' and also has strong fluorescence, and emits intense yellow-gold light when in a lipid-rich environment. Cholesterol esterase (sterol ester thiol hydrolysis) Enzyme, registration number: EC 3.1.1.13) Catalyzed hydrolysis of cholesterol ester and some other sterol esters to release cholesterol and lipids 145179.doc •16- 201109655 Fatty acid anion enzyme. Cholesterol oxidase (cholesterol: oxygen oxidoreductase, Registration No.: EC 1.1.3_6) is an enzyme that catalyzes the oxidation of cholesterol to 4-cholesten-3-one and hydrogen peroxide in the presence of molecular oxygen. Horseradish peroxidase (Sigma Aldrich, registration number: EC m· ?) is a nitrogen peroxide oxidoreductase. Other equivalent hydrogen peroxide reductases are also known to those skilled in the art and are available, for example, from soybeans. For lipid distribution, the first flow path comprises Amplex Red' second flow path Contains K37 and the third flow path contains Nile Red Or the first test area may comprise Amplex Red, the second test area may comprise κ37 and the third test area may comprise Nile Red. Still more preferably, when the first flow path comprises Amplex Red, the first flow path further comprises Cholesterol vinegarase, cholesterol oxidase, and horseradish peroxidase. In an alternate embodiment, the first test region may comprise a cholesterol (tetra) enzyme, a cholesterol oxidase, and a horseradish peroxidase. In still other embodiments The application zone comprises at least one probe, reporter molecule or reagent 'selected from the group consisting of Amplex Red, K37, Nile Red, Cholesterol Esterase, Cholesterol Oxidase or Horseradish Peroxidase. Preferably, the probe The reporter or reagent is in dry form. Advantageously, the apparatus can be used to perform rapid and easy assays (which can be performed simultaneously) to determine the presence/absence or concentration of analytes from biological fluids, for example, amphiphilic polymerization. The use of the material shortens the time taken for the measurement from a few hours or even days to as short as about one minute. In another aspect of the invention, a measurement of water is provided. A method of an analyte in a sample comprising: 145179.doc 201109655 (0 contacting an aqueous biological sample with a combination of at least one hydrophobic luminophore and at least one amphiphilic polymer, wherein the at least one hydrophobic luminescence The group binds to at least one analyte in the aqueous biological sample and emits fluorescence under appropriate excitation when combined with it; (11) exciting the product from step (1) at a suitable excitation wavelength; ((1)) at a suitable detection wavelength Fluorescence emission after step (ii) is tested. Those skilled in the art will appreciate that suitable excitation and emission wavelengths will depend on the particular dye or luminophore used. Standard laboratory techniques or available dyes or luminescence can be used. Group related data to determine the choice of suitable wavelength. In one embodiment of the method of the invention, the aqueous biological sample is contacted with at least one amphiphilic polymer and then contacted with at least one hydrophobic luminophore. In an alternate embodiment, the contacting of the aqueous biological sample with the at least one amphiphilic polymer is carried out substantially simultaneously with its contact with the at least one hydrophobic luminophore. As a non-limiting example, in accordance with the first or second aspect of the invention, a biological sample (in aqueous form) is applied to an application area on, for example, a test wafer or card. The application area is coupled to at least one, preferably three, four or more test areas. The connections between the regions can be achieved by channels, capillaries, obstructions, and the like. Channels, capillaries, and the like are preferably coated with an amphiphilic polymer. When the (four) track is made, it can be formed by printing or coating the amphiphile on the surface. The amphiphilic polymer will take the water sample from the application zone along the channel, capillary, orbit and the like "core 145179.doc •18·201109655 2" or at least one test area. When multiple channels, capillary I, trajectory, and the like are used, the sample can be divided as it travels with the fluid: 2 component fluid. In this way, the sample can be cut A without a manual operation step across the lateral direction of the amphiphilic polymer. Each channel, capillary, obstruction, and the like can comprise a combination of at least one hydrophobic luminophore and an amphiphilic polymer. When present, the hydrophobic luminophores in each channel, capillary, orbital, and the like, may be the same or different. Each of the different hydrophobic luminophores can bind to a particular lipoprotein species or subclass, and when combined with it, alters the fluorescence yield under appropriate excitation, and the fluorescence yield is expressed as the concentration of the lipoprotein species or subclass. Likewise, each channel, capillary or track, and the like can also contain at least one enzyme or other component or reagent. In this manner, when the sample travels or flows (takes) along a channel or the like, other components or reagents are supplied thereto and mixed with other components or reagents without manual operation. Gut, those skilled in the art will appreciate that different amounts or combinations of reagents or conditions can be applied to different sample fractions to perform and measure different assays in each test area. Preferably, at least one hydrophobic luminophore dye, especially a lubricated wood, is more preferred, and more preferably a dye having unique fluorescence when combined with an analyte such as a lipid component.

Human serum albumin (HSA) is an important serum component at a concentration of about 30 50 mg/ml. HSA is known to have at least two binding sites capable of binding to various ligands. The first type is referred to herein as the "hydrophobic domain" and the second domain is referred to herein as the "drug binding domain." Such domains are well known to those skilled in the art and are used in Nature Structura I 145I79.doc 201109655

Biology (V5 p827 (1998)) is different from each other in the paper. This paper identifies that the hydrophobic domain is a domain that binds to fatty acids and that the drug binding domain is capable of binding to many drugs that can associate with HSA. The inventors have determined that a hydrophobic luminophore can bind to the hydrophobic binding site 7 domain of HSA and can fluoresce when bound to HSA. Thus, the inventors believe that this additional fluorescence can cause significant background signals that can distort the measurement and cause errors in lipoprotein concentration measurements. Thus, when the aqueous sample is a blood sample, the hydrophobic binding site of the HSA that binds to the hydrophobic luminescent group is blocked. Therefore, it is preferred to add a ligand binding inhibitor before analyzing the sample. The ligand binding inhibitor can be hydrophobic. The inhibitor can be amphiphilic. The ligand binding inhibitor may comprise a fatty acid or a functional derivative thereof as well as other hydrophobic molecules. Examples of suitable fatty acid derivatives which can block the HSA hydrophobic binding site can include fatty acids, esters, caries, carboxylic anhydrides or guanamines and the like. Preferred fatty acid derivatives are fatty acid esters. The fatty acid or derivative thereof may comprise a C1_C20 fatty acid or a derivative thereof. Preferably, the fatty acid or derivative thereof may comprise a C3_C18 fatty acid or a derivative thereof, more preferably a C5-C14 fatty acid or a derivative thereof, and even more preferably a C7-C9 fatty acid or a derivative thereof. More preferably, the ligand binding inhibitor comprises caprylic acid (C8) or a derivative thereof, for example, an octanoate: preferably, the ligand binding inhibitor is an alkali metal octoate, preferably a Group I alkali metal octanoate (eg , sodium octanoate or octanoic acid If) form added. Preferably, about 1 〇 4 mM of the ligand binding inhibitor is added to the sample prior to analysis, and more preferably about 20-200 rnM of ligand binding inhibition is added 145179.doc • 20· 201109655 ' And even more preferably, about 30-80 mM of a ligand binding inhibitor is added. More preferably, about 50 mM inhibitor is added. Thus, in a preferred embodiment of the method, about 50 mM sodium octanoate can be added to the sample prior to analysis. Ligands of the drug binding domain of HSA include drug molecules such as: scorpion spleen, ibuprofen, diazepam, steroid hormones and derivatives thereof, blood erythroside, Bilirubin, lipophilic prodrug, warfarin, coumarin-based drugs, anesthetics, diazepam, ibuprofen and antidepressants (eg, sputum), benzoic acid or its derivatives (eg tri-benzoic acid or triiodobenzoic acid). Alternatively, the HSA binding domain can be blocked or removed by using an HSA extraction construct (e. g., an anti-HSA antibody). In a particular embodiment, the aqueous sample is used in the "r-state", i.e., the sample is not diluted prior to use. In other embodiments, the aqueous sample can be diluted prior to use. For example, when the aqueous sample is derived from blood, it may be desirable to dilute the sample prior to performing the assay, for example using a 1:80 dilution. Preferably, the diluent is phosphate buffered saline (PBS) and comprises at least one ligand binding inhibitor. In a particular embodiment, the diluent comprises two ligand binding inhibitors, one for the hydrophobic binding site and one for the drug binding site. Preferably, the ligand binding inhibitor comprises benzoic acid and octanoic acid. Alternatively, the sample can be diluted with pBS and at least one ligand binding inhibitor combined with the amphiphilic polymer. Thus, a ligand binding inhibitor is added to the aqueous sample and it is mixed with the aqueous sample as the aqueous sample travels laterally (e.g., along a tube, capillary, channel or track). 145179.doc 201109655 二相 =法中' A two-person or two-fluorescence measurement was performed in parallel under similar conditions.乂 A method for enhancing lateral fluid flow in accordance with a third aspect of the invention, comprising applying a amphiphilic polymer to the surface of the flowable fluid. Preferably, in the method of the invention, the surface is defined by a bureau, a capillary, a well, a membrane, a membrane or the like. It is thus possible, for example, to use an amphiphilic polymer to coat the surface defined by the inside of a tube, capillary & channel, well, membrane or the like. It should be understood that amphiphilic polymers can also be used as a coating for the membrane in a forceful "standard" lateral flow assay to accelerate fluid flow and provide more control over fluid flow. The hydrophobic or amphiphilic polymer can be applied, printed and/or sprayed onto the surface by a printing process (for example by ink jet printing or bubble jet (four) brushing) or other silking methods, for example, forming an obsessive manner" / or layer. In a particular embodiment of the method, the amphiphilic polymer is in the form of a film. In other embodiments, the film may be impaired into particulate material or may form, for example, particles, beads, pellets, microspheres, nanospheres, or microspheres. The at least one amphiphilic polymer can be in the form of particles, beads, pellets, microspheres, nanospheres or microspheres. According to a fourth aspect of the invention, the use of Gafquat for stabilizing proteins, especially enzymes, is provided. Surprisingly, it has been found that a copolymer of 'vinylpyrrolidone and dimethylaminoethyl methacrylate is a suitable enzyme stabilizing agent, for example sold by International Speciality Products under the trade name Gafquat (RTM). . Gafquat (CAS Registry Number: 53633_54-8; 7732-18-5) is a 145179.doc • 11· 201109655 series of water-soluble copolymers (eg, polytetra-ammonium salt _n). It is the main active ingredient of many hair products (such as mousse, gel, hair styling agents) and specialty cosmetics' but has not previously been used to stabilize enzymes or proteins. Although a wide range of enzymes can be stabilized using Gafqua#, preferred enzymes suitable for stabilization with Gafquat include cholesterol vinegar, cholesterol gasification enzyme and horseradish peroxidase. According to a fifth aspect of the invention, there is provided the use of the apparatus, method or process of the invention in the assay. All of the features described herein and/or any of the steps of the method or process disclosed herein may be combined and combined with any of the above aspects, at least some of which Except for combinations where features and/or steps are mutually exclusive. [Embodiment] Referring to Figure i of the drawings, the measuring device ((10) often includes a casing (2a) and a cover (2b) which protects the pure field (3), the flow path (7) and the test area (4) from contamination or damage. The device is equipped with a ridged handle portion (7) that allows a doctor or clinician to hold the device. The outer casing above the application area (3) contains a well (10), thereby having a beveled edge to smash the sample 5 4 π uw The window (4a) is supplied to the entrance of the test area to allow measurement of the measurement results. The outer (2a and 2b) is a hydrophobic plastic strip (6) which serves as a support for the flow path. The flow path (5) is printed onto the cut, in this case from the application area (to the four test areas (4). The filter has just reached the support to filter out the particles in the aqueous sample. 145179.doc • 23- 201109655 In use, the doctor or clinician applies an aqueous sample to the application zone (3). The fluid from the aqueous sample travels along the flow path along the flow path to the filter (8) and through the filter. A filter with pEG prevents any cells in the sample from entering The step travels along the flow path. After the fluid approaches the test area, the knot formed in the flow path divides the sample into four parts. When used to make a lipid distribution, the first liquid separation is carried along the flow path. The K37 dye combined with pEG is supplied to the liquid separation in the flow path and mixed with the Κ37 dye of PEG. The second liquid separation is along the flow path and is mixed with the second dye Nile Red. The liquid is entangled along the flow path 5 (4) and is entangled with it. It is mixed with three kinds of dry enzymes, cholesterol esterase, cholesterol oxidase, horseradish peroxidase and dye Amplex Red. The three samples mixed with the respective dyes are separately flowed. Up to and concentrated in the test area (4). The fourth test area (5d) is used as a control to measure the background glory. The device is loaded into the reader, which in turn excites each test area and measures any subsequent fireflies. (d) Shooting. The processing wafer in the reader calculates the concentration of the analyte (eg total cholesterol, triglyceride, occupational and other lipid components) in the sample and displays the result on the Lcd screen. Figures 2 and 3 show the available For example, measuring blood Other embodiments of the present invention. The present invention is in this embodiment.

. Fork surface (1). The support surface typically comprises or consists of an opaque material, such as a plastic material incorporating a U-material (e.g., carbon black). In this case, the support surface "fluorescent, luminescent or photometrically compatible pharmaceutical grade polymer-type smoker polymers generally have excellent mechanical properties, low spontaneous fluorescent knives 145179.doc •24·201109655 uv transmission. Suitable polymers include cyclic olefin copolymers such as Topas® COC (CAS No. 26007-43-2), Zeonor® COP, Zeonex® COP or Udel® Polyamide (CAS No. 25 13 5-5 1-7) In this example, the branch is formed from TOPAS COC containing 1% carbon black. The use of carbon black has the added advantage of, for example, reduced power requirements during laser welding, increasing the ease of operation and protection of the device. Thermally unstable components in the reaction chamber. The use of carbon black may also have benefits associated with heat dissipation/insulation. The support surface of this embodiment is molded, but may be known by those skilled in the art. - Formed by standard molding or machining techniques. Although it is generally flat, it contains a profiled area (pr〇med), which is formed along the circumference of the application zone (4) in the case of an unken relief. Four (in this case Capillary channel (3). In this case, each of the four channels is spaced apart from each other by an angle of about 9. The molded support surface forms an application zone (4), a fluid flow detection zone (6). This is far from the Yuanjia area (4). The farthest of each end of the π qiu yue is also tapered. The width of the cone is Γ shaped to extend the cross-sectional area of the capillary flow channel. (7) Prevents ingress capillary fluid flow. Defining the force = the scaly end, but can take many other forms. Force = domain, fluid flow path, detection zone The structure of the fine channel is more precisely shown in Figure 4. The hair of the heart should be understood. For example, and # times different shapes to suit specific and as a non-limiting example, the channel can be in the shape of a shape or "tears 145179.doc •25· 201109655 drops" shape, trapezoidal, triangular, round seven π figure "μ 仏 cylindrical Or tube shape. More examples are shown in Figure 4. For hospitals, sub-large volume use, it is conceivable to arrange 1 〇, or 00 capillary channels on the measuring device of the size and shape of the disc. Figure 4 shows a more detailed embodiment of the flow termination knot. In this figure, the fluid flow path (7) along the fluid flow path portion (13) before or after contracting to form a reduced cross-sectional area Essentially all of its length maintains an equal cross-sectional area. This fluid flow path portion is in turn adjacent to and in fluid communication with the chamber (14). The chamber is divided into zones by a low wall or barrier (15) - in this case divided into two parts ( 15a) and (15b). In use, the fluid enters the chamber 〇4) the filling portion ((5)). The wall (15) or more precisely (d) the capillary force of the sample fluid in the wall region prevents the fluid from flowing to the chamber Two parts (15b) t. In other embodiments The wall is replaced by a raised portion (16). Again, the capillary force of the sample fluid acting in the region of the raised portion (16) prevents fluid from filling the entire chamber. Those skilled in the art will recognize that the chamber (14) and wall The configuration (e.g., size, shape, height) of the (15) or raised portion (16) may take a variety of forms including different geometries, such as circular, trapezoidal, and the like. Located in the second or distal region of the chamber (15b) The nearby vents allow air to move from the chamber to the atmosphere to balance the pressure within the fluid flow path of the device. The support surface also includes a plurality of alignment posts (8) to align the support surface with the second surface-covering member (2) The covering member (2) is also formed from a suitable pharmaceutical grade polymer (for example, as described above). In this example, 'the cyclic olefin copolymer (Topas C〇C) is used again (CAS No. 26007-43- 2), but in this case no carbon black is used. I45179.doc • 26- 201109655 • At least for the determination of the reader, therefore, the covering part is optically clear/transparent excitation/emission wavelength. Covering part (2) also Molded, and slinged It is flat and contains a plurality of holes. The cover member can be a branch member. The Ba includes a gasket or a spacer between the two. The hole (9) is aligned with the w-six and allows the user to apply the area. A fluid sample is applied. A small hole in the water and eve series is used as the vent (10). In the non-embodiment, the aligning hole (1) is positioned relative to the baffle member by accommodating the alignment post (8). In the #代#, the hole and the alignment hole are provided in the supporting member, and the covering member only plays the covering function. The aligned 7L member can be simply the protruding member and the corresponding alignment hole or groove. The supporting member and the covering portion The components may be secured or joined together by, for example, mechanical means (e.g., by screws, rivets, bolts, or tabs). Alternatively, the support member and the cover member may be joined together by a friction fit. In other embodiments, the cover member and the support member are joined together by, for example, using glue, solvent, tape, and the like. In this case, the cover and the deck components are joined together by using heat welding and laser welding. One or both of the support member and the cover member may comprise an identification member to provide a unique identification and/or to provide a means for the assay reader to, for example, monitor the number of uses of the assay device or the application time of the sample to survive Determination time. The identification members may also contain instructions or data such as calibration or quality control data. The identification member can take a variety of forms including, but not limited to, text, blind braille, digital data, one-dimensional bar codes, two-dimensional bars, 145179.doc -27-201109655 code, RFID tags, and the like. The transition device (12) is held securely between the branch member and the cover member adjacent to and in fluid communication with the application region (4) and the aperture (9) and the fluid flow path. In use, the undiluted or diluted aqueous sample is applied directly to the filter via a hole (9). The fluid in the sample moves through the filter and, as such, larger particles are removed by passive filtration. In the case of whole blood, the particles include red blood cells. This reduces background interference caused by such larger particles. When blood (e.g., whole blood, venous blood) is used, the filter may further comprise an HSA extraction member comprising an anti-HSA antibody that removes hsa from the sample as described above, again reducing the background. Blood can be obtained by finger pricking or by heel puncture in the case of a newborn. This is the way to open a small wound, such as at the fingertip, which produces no more than a few drops of blood (about less than 5 〇 μ1, such as about 1 〇 μΐ to 20 μ1). After the blood droplets have formed, they can be applied directly to the application area of the assay device or by suction through a pipette and subsequently applied accordingly. Other ways of obtaining blood samples are known to those skilled in the art. The ability to use the blood obtained by, for example, finger pricking directly represents a significant advantage over assays well known to those skilled in the art. Alternatively, an assay device can be used to test a sample derived from plasma, in which case the sample can be diluted 1:80. Typically, the diluent used is phosphate buffered saline (PBS) and it can comprise at least one Body binding inhibitors. The diluent may also comprise two ligand binding inhibitors, one for the hydrophobic binding site and one for the drug binding site. The fluid travels from the application zone (4) by means of capillary flow into the gripper w-path (5) in communication with the application zone 145179.doc -28- 201109655. As it travels, the liquid combines the dried reagents (e.g., fluorescent dyes and enzymes) with water. Therefore, the sample is moved by capillary or lateral flow, and no external force such as *pump is required to move the aqueous sample. Amphiphilic or nonionic polymers enhance capillary flow and reagent mixing efficiency. The use of the "5" form means that the component remains generally free of or empty in the form of liquid or moisture; that is, it is reconstituted by the implementation of the crucible itself rather than prior to the testing procedure and independent of The assay procedure is reconstituted in solution form. The aqueous sample itself reconstitutes the dry agent, thereby eliminating the need for separate reconstitution buffers and steps. As noted above, the use of an amphiphilic or nonionic polymer facilitates the mixing of the dry reagent with the aqueous sample. When an enzyme or other reagent is used, it is preferred to stabilize the enzyme or other reagents, especially when used in a dry form. In the above-mentioned t, the "stabilized reagent" is a reagent having improved stability such as storage stability, thermal stability and the like. Therefore, in the specific implementation, the reagent package 3 used is a stabilizer. A specific method of stabilization is disclosed in the International Patent Application No. PCT/A No. 5,182, the disclosure of which is incorporated herein by reference. Other suitable stabilizers include copolymers of vinylpyrrolidone and dimethylaminoethyl methacrylate, for example by

International Speciality Products is available under the trade name Gafquat (RTM). GafqUat (CAS Registry Number: 53633-54-8; 7732-18-5) is the name for a series of water-soluble copolymers (e.g., polytetra-ammonium salt _ 1 i ). As the front fluid moves forward, an equal volume of air is expelled through the vents, and the pressure within the device is balanced. After the fluid reaches the flow termination junction, the surface tension resistance 145I79.doc -29· 201109655 stops the capillary flow. At this stage, the device can be placed in a suitable assay reader and the amount of analyte (e.g., cholesterol and blood lipids) measured. The housing of the assay device is typically adjusted to be placed in communication with the helium reader. For example, the assay device can be inserted into a reader, placed on a reader, or attached to a reader, and the reader can include a station member (eg, a socket) or an alignment member to enable the assay device to be appropriate Insert, place or attach. In this embodiment, the covering member of the measuring device has a "v" shape. This helps the device to align with the reader. Typically, the measuring device of the present invention is disposable, but the reader is typically reusable. The assay device can be used in a variety of methods or reactions, such as immunoassays and luminescence assays, including cholesterol, lipoprotein or triglyceride assays. Immunoassays are biochemical tests that measure the concentration of a substance in an aqueous sample, such as serum or urine. This assay utilizes the specific binding of an antibody to its antigen by the reaction of the antibody with its antigen. Monoclonal antibodies are preferred because they bind to a specific molecule and provide specific and accurate testing. The presence of both antigens or antibodies can be measured, for example, when an infection is detected, the presence of antibodies against the pathogen can be measured. Alternatively, hormone biomolecules can be used as antigens when measuring biomolecules such as hormones and the like. The response of the measured aqueous fluid can be compared to a standard of known concentration, for example, a standard curve is plotted on the graph. Detection of the amount of antibody or antigen can be accomplished by a variety of methods, such as labeling an antigen or antibody. As a limiting example, the label may consist of an enzyme (EIAst ELISA) 'radioisotope (e.g., I.125), a magnetic label, or a luminescent or fluorescent label. Advantageously, the apparatus of the present invention relies on capillary flow to effect fluid transfer of low volume samples by using amphiphilic or nonionic polymers, and thus does not require the use of moving parts. Therefore, the device overcomes the problems of scale, economy, manufacturing, and mechanical deficiencies encountered in the prior art. The assay reader for use with the assay device of the present invention may be adapted to receive two or three assay devices, e.g., from multiple patients or for multiple assays of aqueous samples from individual patients. The reader can include two (or more) excitation members that can be aligned with the detection zone of the assay device. The "exciting member" is operable to excite the sample under test, for example, to fluoresce it. The apparatus also includes at least one detection member operable to detect, for example, fluorescent light emitted by the sample in the detection zone. Typically the 'excitation member' comprises an illumination source operable to illuminate the sample at about 4 〇〇 nm to 600 nm. Therefore, the light source is preferably capable of illuminating the sample at about 4 〇〇 nm_6 〇〇 nm. The illumination source can include one or more light bulbs, or one or more LEDs, or other sources (eg, one or more lasers). The excitation wavelength can be varied using at least one interference filter. The excitation member can also include a polarizing member operable to polarize light generated by the illumination source. The excitation member may further comprise a focusing member adapted to focus the light onto the sample. The focusing member can comprise a lens or light guide, such as a fiber optic or optical film (3M). The detection member may comprise a photodiode, a CCD, or a photomultiplier tube or an optical sensor (which is preferably sensitive to yellow-red). The fluorescent end of the sample emission may be from about 440 nm to 650 nm. Detected within the range. The detection member should be capable of detecting fluorescence emitted at about 490 nm, about 495 nm, about 570 nm, about 600 nm, and about 610 nm. Fluorescence can be collected by the second lens 145179.doc •31 - 201109655 and can pass through the polarizer. The scattered excitation light can be removed by a cut-off transition or a belt pass filter. When measuring the fluorescence intensity, the current from the photodiode or the count rate from the photomultiplier can be from the current meter, voltmeter or count rate. The meter module reads out. Those skilled in the art will be familiar with other methods. The reader may also include an excitation correction system to account for fluctuations in the source. The apparatus can include at least one fluorescent standard for calibration prior to determining the analyte concentration. The standard can be an internal standard. In a particular embodiment, the assay reader is configured to detect and measure the fluorescence intensity of a single or a plurality of assays simultaneously or sequentially as the assay device enters the reader or thereafter. The reader may also include a processing member adapted to determine the concentration of the analyte in the sample based on the detected fluorescence. The reader may further comprise means for displaying the measured value determined from the sample

The display member is preferably in readout form. For example, the display member can include an LCD screen or can be powered and/or calculated and/or displayed depending on the computer. In its most basic form, the display member can simply be a window that displays indicator readings or measurements. Typically, the assay reader is portable and, advantageously, the assay device and the reader can be used to perform assays simply, quickly and simultaneously to determine the presence/absence of an analyte of an aqueous sample, such as a biological fluid, or concentration. For example, a clinician with knowledge of cholesterol, lipoprotein, and progenitor concentration can use the device to determine an effective treatment procedure. In addition, the measuring device and the reader are portable and can be used by a nurse in the back or out of the clinic, or even in the form of a test kit for home use. I45179.doc • 32· 201109655 In particular, the treatment member is adapted to directly determine the concentration of an analyte such as cholesterol, triglyceride, hdl, ldl, and IDL, or a plurality of analytes based on fluorescence analysis. Alternatively, the treatment member can be adapted to calculate the concentrations of LDL, VLDL and IDL in the sample based on the concentration of total lipid, cholesterol, alcohol and HDL. Use of the device in cholesterol and lipid analysis The following examples illustrate the use of the assay device of the present invention to measure lipoproteins in aqueous biological samples. In this example, the fluid flow path is coated with an amphiphilic polymer pEG to accelerate fluid transport. For lipid distribution, the amphiphilic polymer is also combined with a fluorescent dye such as Amplex Red, K37 or Nile Red and/or other reagents such as enzymes, coated or printed in or within the fluid flow path. Fluorescent dyes and/or other reagents can be printed in the form of one or more picolite liter droplet arrays. Suitably, the array can comprise from about 15 to 4500 droplets along the first axis multiplied by 25 to 1 droplets along the second axis. Specific array sizes include approximately 34 x x 65 droplets per mm 2 , approximately 3000 χ 65 droplets, approximately 1500 X 65 droplets, approximately 19 x x 65 droplets, 600 χ 65 droplets, 450 χ 65 droplets or about 4 x x 65 droplets. Those skilled in the art will appreciate that variations in the density of these droplets have minimal impact on the performance of the assay, but it should also be understood that, conversely, the densities can be optimized to improve assay performance. For example, the droplets may also be applied in the form of nanoliters or femtoliter droplets, applied in an overlapping array, applied in the form of one (or more) ones (or more), separated at intervals. The discrete individual arrays are applied or applied in the form of blocks (ie, cumulative) of a plurality of arrays 145179.doc-33-201109655 forming a larger array. The array size can also be optimized for, for example, a particular reagent or dye concentration. When "printing", the light dye droplets can be applied to the device at a concentration of about 3 to 3, more suitably 3 to 2.5, and even more suitably about 5 to 2.0. . Alternatively, the concentration of the fluorescent dye can range from about can to thin when the device is used to dilute a blood sample. The available concentration of the fluorescent dye is 彳(四)福, which is used in undiluted blood samples; the agricultural system is about 2. mM. @后干燥染料, then use 6 when the sample fluid combines the amphiphilic polymer water to dilute the lab light dye, which in turn can bind to the lipoprotein in the sample. When so combined, the dye fluoresces under appropriate excitation. The total lipoprotein concentration in the sample was determined by fluorescence analysis. The method generally comprises: (i) contacting an aqueous biological sample with at least one dye or luminophore and at least one amphiphilic polymer, wherein the at least one dye or luminophore binds to at least one lipoprotein in the aqueous biological sample, And emitting fluorescence under appropriate excitation when combined with it; (11) exciting the product from step (1) at an excitation wavelength between about 400 nm and 620 nm; (iii) at a wavelength between about 440 nm and 65 nm Fluorescence emission after the measurement step (ϋ). This method can be used to make lipid profiles for aqueous biological samples. The term "total lipoprotein" as used herein means the sum of the concentrations of triglyceride and total cholesterol in at least VLDL, HDL, LDL, 145179.doc -34 - 201109655 IDL and the total acne length of the chylomicron 'in other words' sample. . The term "total cholesterol" as used in the field means the total concentration of cholesterol in the sample. The fraction used in the "solution of juice" § "lipid distribution" means the concentration or relative concentration of the lipid component (ie, total lipoprotein and 绰膪, ", cholesterol and triglyceride) in the sample. ^ Most lipids in blood or serum samples bind to lipoproteins. The clinical trials to the Zhongshi & $ test did not measure total lipoprotein. Therefore, it is customary to first determine the concentration of cholesterol and cholesterol ester and the concentration of triglyceride vinegar and then add them to determine the total lipoprotein concentration. There is considerable error in the amount of monoglyceride g in the clinical laboratory because it relies on the measurement of natural circulating glycerol in the blood. Advantageously, the total lipoprotein concentration (equal to total lipid concentration) is determined by directly measuring the amount (volume) of lipoprotein particles, so the cholesterol assay of the present invention does not have this error. Therefore, an error such as a concentration of triglyceride caused by circulating glycerin in the sample can be avoided. In the first international patent application PCT/GB2005/004757 (published in WO2006/061646), the present inventors propose a simplified assay based on the use of, for example, K37 to measure lipoproteins in biomacromolecules, which are desired by clinicians to be rapid and It is especially useful in the case where the lipid distribution is effectively obtained. For the determination of the concentration of total lipoproteins (ie, hdl, LDL, IDL, and VLDL) in blood samples using K3 7 fluorescence measurements, the inventors recognized that for a given total lipoprotein concentration, binding to each lipoprotein The fluorescence response of the species K3 7 (ie, 'total lipoprotein concentration) may be substantially the same regardless of its composition (ie, the ratio in the sample). Therefore, the best case is 145179.doc -35- 201109655 = use K37 in the following way: the fluorescence intensity response is substantially unrestricted in the range of the expected molecular weight of the expected lipoprotein in the clinical test. Based on any assumptions, we believe that the intensity of fluorescence from fluorescent dyes depends on their affinity for specific lipoprotein molecules (HDL, LDL, IDL or) in the sample. The quantum yield of fluorescence depends on the environment within the lipoprotein molecular complex and the degree of fluorescence quenching caused by energy transfer between closely packed probe molecules. Thus, in the prior application, the present invention may be selected for use with suitable probe species concentrations and excitation and emission wavelengths for accurate total lipoprotein measurements by simple fluorescence measurements. The inventors have progressed. It is forbidden that 'because the probe has a higher affinity for HDI^, the probe concentration can better balance 7 of the hdl compared with VLDL and LDL, and thereby balance HDL. The higher degree of quenching within is such that all lipoprotein particles produce a constant fluorescent signal response. The inventors performed a series of experiments to investigate whether the fluorescent material of the probe substance K37 and the type of each lipoprotein particle (hdl, [〇1^ and lipoprotein concentration (which may be encountered in actual serum clinical samples) can be obtained. Linearity and equivalence between lipoprotein concentrations. Surprisingly, 'they found a linear relationship between the fluorescence of K37 and the concentration of lipoprotein at a specific K3 7 concentration and specific excitation and emission wavelengths. Using the method of the prior patent application (PCT/GB2005/004757), those skilled in the art can recognize other suitable dyes which also exhibit the relationship with the concentration of lipoprotein. It is advantageous to use the enzyme in the determination of cholesterol. Cholesterol is usually found in an esterified state, so cholesterol esterase is preferably used to hydrolyze cholesterol ester 145179.doc -36 - 201109655 to release cholesterol. The free cholesterol sterol can then be converted to cholesterol by the action of cholesterol oxidase - 4-en-3-one, which produces perylene peroxide in the process. Advantageously, 'Amplex Red and hydrogen peroxide are converted to resorufin and water by horseradish peroxidase. It can be detected as a fluorescent compound whose maximum absorption is at about 563 nm and the peak emission wavelength is 587 nm. The total cholesterol content can be obtained by exciting the sample at about 485 nm and measuring the fluorescence at about 600 nm. For example, when an enzyme is used, it can be provided in excess of the ratio used to measure, for example, the enzyme Km many times. This ratio can be very high compared to the approximately 1:1 ratio used in the industry. Surprisingly, the difference in surface energy of the device plus the use of a combination of dry stabilized enzymes and optionally amphiphilic polymers allows the use of very small amounts of samples and reagents. Without limiting the theory, we believe that at least two The difference in surface energy between partially opposing surfaces creates a circular motion in the laminar flow of the aqueous sample, resulting in more efficient mixing. Due to this increased efficiency, a larger amount of enzyme can be used in a smaller reaction volume, thereby achieving a prior art The previously achievable reaction of the method is more efficient and rapid. The second step of the method may comprise: (ii) exciting from step (1) at an excitation wavelength between about 400 nm and 520 nm. The excitation wavelength can be from about 420 nm to 480 nm or from about 440 nm to 470 nm. The excitation wavelength used depends on the specific fluorescent dye used in the assay. For Amplex red, the excitation wavelength is about 48 〇 nm; for K37, the excitation The wavelength is about 440 nm; and for Nile Red, the excitation wavelength is about 58 〇 nm. 145179.doc •37· 201109655 The third step of the method consists of: (iii) the amount at a wavelength between about 490 nm and 650 nm. Fluorescence emission is measured. Alternatively, the fluorescence emission can be measured at a wavelength of approximately 520 nm to 620 nm. At an emission wavelength of about 540 nm or higher, a more accurate reading for determining the total lipoprotein concentration (i.e., the concentrations of HDL, IDL, LDL, and VLDL, and, if present, the concentration of the chylomicrons), can be observed. However, the preferred fluorescence emission wavelength measured depends on the particular fluorescent dye used in the assay. for

Amplex Red, the Luguang system is measured at about 600 nm; for K37, the fluorescence is measured at about 495 nm; and for Nile Red, the fluorescence is measured at about 61 〇 nm. And the emission wavelength does not need to be measured at the optimum wavelength of each specific dye. It is optional to give the optimum separation or performance wavelength when the dye is used in combination or in parallel (e.g., when the assay is performed simultaneously in a single assay device). It should be understood that step (ii) and excitation and detection can also be carried out substantially simultaneously. Alternatively, the concentration of triglyceride can be calculated by subtracting the total cholesterol concentration from the total lipoprotein concentration. Thus, a more detailed lipid profile of the sample consisting of total melatonin concentration, total cholesterol concentration, and triglyceride concentration useful to the clinician is thereby produced. The inventors have previously discovered that many dyes bind to lipoproteins and exhibit different fluorescent responses depending on the particular lipoprotein bound. Fluorescence spectrometry of these dyes can distinguish between the various types of lipoproteins present in the sample. This is achieved by enhancing or attenuating the fluorescence caused by a lipoprotein in a lipoprotein mixture with fluorescence expected from other lipoproteins (in the absence of a specific propped lipid 145179.doc -38 - 201109655 protein) Comparisons were performed to determine 'as determined by a calibration curve from total lipoprotein content and known values. For example, the fluorescent dye Nile Red exhibits significantly higher fluorescence in hdl than in other lipoproteins such as LDL and VLDL. Therefore, other fluorescent dyes (such as Nile Red, K37 or any other lipophilic probe that exhibits specificity for a particular lipoprotein or enhanced or attenuated by fluorescence) can be used to distinguish lipoprotein species or subclasses in a sample. . Thus, the method of the invention enables the use of fluorescence analysis to determine the concentration of a particular lipoprotein species or subclass in a sample. Typically, this involves the use of a second and/or third fluorescent dye to determine the concentration of a particular lipoprotein species or subclass by shifting the fluorescence response of a dye specific for lipoproteins. As an example, the calculation is performed on the excess fluorescence emitted by the presence of HDL by the use of Nile Red to determine the HDL concentration in the sample. First, the total lipoprotein concentration (measured as Γ Α) was measured by a linear relationship between Κ37 fluorescence and lipoprotein concentration (as measured by step (1)). Second, LDL (and/or VLDL is used, since the fluorescence versus concentration response must be essentially the same). Nile red fluorescence is calibrated at different concentrations to obtain a calibration curve with a slope of "χ" and a intercept of "Υ". Those skilled in the art will know how to prepare a range of concentrations of LDL (and/or VLDL) and determine the corresponding fluorescence for each concentration. A calibration curve of a series of concentrations of HDL and a constant concentration of LDL· can be constructed to obtain a slope "Z". The total lipoprotein concentration measurement "A" (known from K37) and the excess Nile red fluorescence "B" of the unknown sample are known, and the concentration "H" of the HDL in the unknown sample can be determined by the following formula: Y) )/Z. 145179.doc -39- 201109655 It should be understood that pre-made calibration curves or standard calibration curves can be used in practice. Furthermore, the assay device of the invention or the assay reader for use with the devices developed to obtain a lipid profile may comprise internal standards and/or have a treatment that allows for the automatic calculation of lipoprotein concentrations without user intervention. member. Therefore, it should be understood that fluorescence measurements can be used to determine HDL, VLDL (by calculation), LDL (by calculation), total lipoprotein 'triglyceride (by calculation), and total cholesterol concentration. All of these parameters can be measured simultaneously in parallel by exciting and measuring glory over a similar wavelength range. As discussed above, this is a significant improvement over conventional assays, which must be performed separately and typically in a dedicated laboratory, resulting in delayed results. In addition, the fact that multiple lipid parameters can be measured simultaneously greatly simplifies the instrumentation required to perform the measurements. The resulting lipid profile includes determining the concentration of cholesterol that binds to LDL in the sample. Since LDL cholesterol is highly atherogenic, it is particularly advantageous to know the concentration of LDL cholesterol. Thus, the method provides for multiple reads of at least three, preferably four or five, or more parameters of the lipid composition of the sample. Further, the concentration of cholesterol_VLDL-cholesterol can be calculated/evaluated from the concentration of triglyceride, since it is generally assumed that most of the triglycerides are carried in VLDL and the cholesterol component of VLDL accounts for 2%. This is especially beneficial for helping clinicians decide the appropriate treatment process. EXAMPLES The invention will now be illustrated with reference to the following examples, which illustrate the full description of the embodiments herein. 145179.doc -40· 201109655 Example 1 Total Cholesterol Determination This assay uses a triple enzyme system capable of converting a cholesterol or cholesterol ester molecule into a cerium oxide (H2O2). The resulting hydrogen peroxide is then used to oxidize the dye Amplex Red (without fluorescence) to produce a highly fluorescent product, resorufin. Stabilization of the enzyme to plastics The following enzymes and dyes were used for the determination of total cholesterol: cholesterol esterase (3.1.1.13) cholesterol oxidase (1.1.3·6) horseradish peroxidase (1.11.1.7)

Amplex Red : 10-Ethyl _3,7-dihydroxyphenoxazine Stabilize the enzyme onto the plastic using Gafquat as a stabilizer. Three enzymes were added to a 0.01 Μ potassium phosphate buffer solution (pH 7 〇). The final activity of each enzyme was measured as 200 U/ml buffer. Then use the solution

The Gafquat (polymer with a large amount of positive charge) was diluted 1:1 and the resulting solution was deposited onto the plastic surface and dried at 3 Torr for 2 hours in the presence of silicone. This process yields a dried enzyme biosurface with 0.5 U of various enzymes deposited. Assay Procedure - Two passages were taken using 1/80 sample dilutions: a) reacting cholesterol samples (plasma) with dry enzymes and Amplex Red in solution; b) stabilizing cholesterol samples with dried enzymes and drying The Amplex Red dye reaction: (a) The cholesterol sample (plasma) reacts with the dry enzyme and the Amplex ruler in solution. 145179.doc •41 · 201109655 Dilution Buffer A: 4.16 mM Amplex Red, l〇 mM biliary, 〇·2% Triton X-100 is stored in Dulbecco's phosphate buffered saline (pH 72). First, the sample to be determined is diluted in the dilution buffer A in an amount of 80 parts, and then the 5 μM diluted sample is used to reconstitute and activate the dried triple enzyme mixture in the sample measuring chamber (pre-stabilization as described above) ). The cholesterol content was measured by exciting the sample at 480 nm and measuring the fluorescence at 600 nm. The cholesterol concentration was measured directly by measuring steady-state fluorescence or Vmax (maximum yield generation rate) after 4 sec. Each evaluation is carried out by referring to the measurement standard data. (b) Cholesterol s pattern (investigation) and drying enzyme and Ampiex Re (j reaction. Dilution buffer B: 1 mM cholesteric acid, 0.21⁄4 Triton X-ioo in 〇11 6 (^ 〇 酸盐 salt Buffered saline (1^7.2). The procedure is similar to the above procedure. However, in this process, the Amplex Red dye is dried in the flow path with the triple enzyme mixture. First, define one of the assay chambers as described above. The area is coated with 〇·5 u cholesterol from chymase (3.1.1.13), 〇.5 1; cholesterol oxidase (1.136) and 〇.51; horseradish peroxidase (1. Π.丨7). The second and separate areas of the assay chamber were coated with 10 μl AmPlex Red / PEG 2000 solution and dried in the presence of tannin at 3 ° C for 2 hr. The dye coating solution was stored in dimethyl nitrous (DMS0). 5.5 mg/ml Amplex Red, 5% w/v PEG2000. Dilute the sample to be diluted in 1 part to 8 parts in dilution buffer B, and use 50 μM to dilute the sample in the sample measurement chamber. Reconstruction and activation of dry 145179.doc •42- 201109655

Amplex Red dye and triple enzyme mixture. The cholesterol content was measured by exciting the sample at 480 nm and measuring the fluorescence at 6 〇〇 nm. The cholesterol concentration was again measured directly by measuring Vmax (maximum rate of production) or steady-state fluorescence after 40 seconds. The evaluations were carried out by reference to the measurement standard data. It has been determined that both assays are capable of determining 2-11 clinically relevant cholesterol levels. Assay Procedure - Use of Undiluted Samples Pure biological samples are determined by applying the sample to a borosilicate filter infused with an anti-HSA antibody in a consumable device for transition And direct the sample to the appropriate 2 pm deep reading or detection area. The reading area is first pre-coated with 400 χ65 enzyme reagent microliter liters per mm2, followed by 600 χ65 dye reagent microliter droplets per mm2 and 450 χ65 inhibitor reagent micro-liter droplets per mm2. Helps to quickly flow the sample into the reading area. The sample is then excited at 480 nm (10 nm bandpass) to produce fluorescence, which can be detected by a 600 nm (10 nm bandpass) filter to allow for the determination of the total sample by reference to an appropriate standard measurement. Cholesterol content. Enzyme reagent: cholesterol esterase, cholesterol oxidase and horseradish peroxidase were each dissolved in a Gafquat stabilizer mixture at 200 units/ml. Detergent solution: Dissolve 1.63 g of cholic acid, 1 〇g of polyethylene glycol 2 〇〇〇, 800 μΐ of Triton X-100 and 25 3 ·3 μ of diethyl maleate in dimethyl decylamine (DMF) ) and adjust the final volume to 4 〇 ml. Dye Reagent: Add 5 mg Ampliflu Red solid to 480 μΐ Wash 145179.doc •43- 201109655 solution. Inhibitor Reagent: Dissolve 260 potassium trihydrate in water Example 2 - Total Lipid Determination mg sodium azide and 912.92 mg acid hydride, and the final volume was adjusted to 40 ml. The K37 dye was dissolved in DMF to give a final concentration of 〇. Then, 5% w/v PEG 2000 was dissolved in the dye solution and (9) liter of the resulting solution was deposited onto the plastic surface, and dried in the dark at room temperature for 1 hour in the vacuum by removing the solvent. Measurement procedure - use of diluted sample First, the sample to be measured (plasma) is taken! The fraction was diluted to 8 parts in phosphate buffered saline (pH 74) containing 5 mM sodium octanoate. A 5 μl diluted plasma sample was applied to the dried dye, which spontaneously hydrated. The total lipid content was measured by exciting the sample at 440 nm (1 〇 band pass) and measuring the fluorescence obtained through a 495 nm filter (10 nm band pass). The total lipid content was determined empirically by reference to known standards. Assay Procedure - Use of Undiluted Samples A pure biological sample is determined by applying the sample to a borosilicate filter infused with an anti-HS A antibody in a consumable device for use in a filter. Filter and direct the sample to the appropriate 2 〇〇μηη deep reading area. Pre-coating the reading area per mm2 of 3 χ65 2 mM K37/5% (w/v) PEG2000 picoliter droplets in DMF' This helps to quickly flow the sample to the reading area Medium, and the dye is spontaneously distributed into the lipoprotein contained in the sample. The sample is then excited at 440 nm (10 nm bandpass) to generate 'fluorescence' which can be detected by a 495 nm (10 nm bandpass) filter, with 145179.doc • 44 - 201109655 allowed by reference to the appropriate standard amount The total lipid content of the sample was determined. Example 3 - HDL Cholesterol Determination Nile Red was dissolved in DMF to a final concentration of 〇 5 mM. Then, 5% w/v PEG 2000 was dissolved in the dye solution, and 6 liters of the resulting solution was deposited onto the surface of the plastic, and dried in the dark at room temperature for 1 hour in the vacuum by removing the solvent. Assay procedure - use of diluted sample First, the sample to be tested (plasma) was diluted in 1 part to 80 parts in phosphate buffered saline (pH 7.4) containing 50 mM sodium octanoate. A 5 μl diluted blood sample was applied to the dried dye, which spontaneously hydrated. The HDL cholesterol content was measured by exciting the sample at 58 〇 nm (1 〇 band pass) and measuring the fluorescence obtained by a 610 nm filter (1 〇 band pass). The HDL cholesterol content is calculated by using the algorithm set forth in the main description. Equivalent assays can also be performed using undiluted whole blood. Assay Procedure - Use of Undiluted Samples A pure biological sample is determined by applying the sample to a borosilicate filter infused with an anti-HS A antibody in a consumable device for use in a filter. Filter and direct the sample to the appropriate 2 〇〇μηι deep reading area. The reading area was pre-coated with 3400 χ65 micro-liter droplets of 0.5 mM Nile Red/5% (w/v) PEG2000 in DMF, which helped to quickly flow the sample into the reading area. And the dye is spontaneously distributed into the lipoprotein contained in the sample. The sample is then excited at 58 〇 nm (10 nm bandpass) to generate 'fluorescence' which can be detected by a 610 nm (10 nm bandpass) transitioner to allow for the determination of the sample by reference to an appropriate standard measurement. HDL-c contains 145179.doc •45· 201109655 quantities. The data from the three tests set forth in Examples 1, 2 and 3 were processed to provide the following results: Total cholesterol measurement - ie test (1) Total lipid concentration measurement - ie test (2) HDL Cholesterol measurement - ie test (3) Triglyceride calculation - ie test (2) minus (1) VLDL · calculated value - ie the value of triglyceride / 2.2 LDL calculated - ie Determination by Formula Friedwald Example 4 - Use of PEG to enhance the lateral fluid flow in a near-horizontal capillary 100 mm long and with a 2 mm, 1 mm and 0-5 mm internal diameter glass capillary remains untreated, detergent treated or coated Apply peg. The stripper-treated capillary was prepared by washing with virkon and Triton X100 5% solution followed by drying. The PEG-treated capillary was prepared by flowing 5% (w/v) PEG present in the gas sample through the capillary and allowing the excess to drain after drying. The treated and untreated capillaries are held in a near horizontal position (about 1 Torr. Upflow angle) and the tip of the capillary is immersed in water. Measure the travel distance and flow rate of the water moving into each capillary: Untreated capillary: 2 mm _ reaches 2〇mm 1 mm in about 30 seconds - reaches 9〇mm in 15 seconds 〇.5mm • at 18 The tube reaches the end of the tube (100 mm). The detergent-treated capillary 145l79.doc •46· 201109655 2 mm - reaches 20 mm in about 20 seconds 1 mm - reaches 90 mm in 12 seconds 0.5 mm - within 15 seconds At the end of the tube (100 mm) Capillary coated with PEG: 2 mm - reaches 80 mm in about 20 seconds 1 mm - reaches the end 0.5 mm in 1-2 seconds - reaches the end of the tube in 1-2 seconds (1 〇〇mm) This data shows that the capillary fluid flow rate in a capillary coated with PEG is about four times that of a detergent-treated capillary and about six times that of an untreated capillary. The capillary is rendered hydrophobic by treatment with a deuterating agent (dimethyldichloromethane) and roasting at 12 (rc). Water does not enter the lumen of the hair cells. The hydrophobic capillary is coated with PEG (as above) The fluid flow condition is restored to the same level as the capillary coated with PEG without sibaat j〇n. In some experiments where the peg surface coating is discontinuous, the fluid flow stops at the pE (} coating discontinuation) Experiment 5 - Use peg to enhance flow in vertical capillary tubes Treat capillary tubes of length mm and 2 mm, 1 mm or 0.5 mm in diameter as described above. Fix the capillary in a vertical position and tip the capillary Immerse in water. Measure the height of the water in the vertical capillary: Untreated tube: 2 mm diameter -9 mm 1 mm diameter -22 mm 145179.doc 47- 201109655 0.5 mm diameter -51 mm Detergent treated tube 2 mm diameter - 10 mm 1 mm diameter - 22 mm 0.5 mm diameter - 53 mm PEG coated tube 2 mm diameter -11 mm 1 mm diameter - 25 mm 0.5 mm diameter - 54 mm water does not enter hydrophobic yttrium Capillary. Capillary flow by applying PEG to a capillary containing ruthenium The height is restored and is close to the PEG-free capillaries containing no ruthenium. Use the formula h=2yCos0/pgr (where h is the height (m); γ-system surface tension; lanthanide contact angle; ρ-system density; g The acceleration caused by gravity; and the radius of the r-tube (m)) is calculated from the theoretical maximum height at sea level · 1 4 mm - diameter 2 mm 28 mm - diameter 1 mm 56 mm - diameter 0.5 Mm Example 6 - Transfer reproducibility and long-term stability The following is a measurement of the transfer reproducibility and long-term stability of a hydrophobic molecule (ie dye) / amphiphilic polymer mixture: Dissolving PEG having a molecular weight of 2000 Da in a hydrophobic dye (ni Luohong or K37) is stored in dimethylformamide (DMF) at a concentration of 5% w/v. PEG/dye film is a PEG/dye solution by storing 25 μM in DMF 145179.doc -48- 201109655 was deposited in a 5 ml glass vial. The solution was allowed to diffuse at the bottom of the vial and then placed in a vacuum chamber for 1 hour to evaporate the solvent. Transfer reproducibility was added to the lipoprotein solution by comparison to DMF. The dye of the dye has the same fluorescence intensity as the redissolved dye/PEG film The fluorescence intensity of the protein solution was measured. The reproducibility was calculated by obtaining the coefficient of variation (CVs) of the fluorescence intensity of the 10 dye/PEG films present in the lipoprotein solution. The stability was evaluated by the following • Coating the film for long-term storage and measuring the fluorescence intensity of the films dissolved in the lipoprotein solution after different storage times. The film was stored in the dark under a range of conditions: no desiccant in air, silicone in air and silicone in vacuum. The film was stored under the conditions of 20 ° C and 37 ° C. Determination of the fluorescence intensity of the dye dissolved in DMF or in the lipoprotein solution in the PEG film: K37 Nile Red from DMF 268000 297000 Fluorescence intensity reading from PEG film 269000 296000 PEG film added and dissolved in DMF The fluorescence intensity readings obtained for the dyes in the middle are the same (within 0.5%). Calculate the reproducibility of fluorescence readings of lipoprotein-containing dyes/PEG films: K37 Nile Red Film 1 6.23E+05 4.27E+05 Film 2 6.25E+05 4.27E+05 Film 3 6.24E+05 4.29E+ 05 Film 4 6.25E+05 4.26E+05 Film 5 6.24E+05 4.27E+05 145179.doc -49· 201109655 Film 6 6.26E+05 4.30E+05 Film 7 6.23E+05 4.30E+05 Film 8 6.25E+05 4.28E+05 Film 9 6.22E+05 4.28E+05 Film 10 6.22E+05 4.26E+05 Average 6.24E+05 4.28E+05 SD 1370.32 1475.73 CV (%) 0.22 0.34 The dye has a CV of less than 0.5%. Stability measurement time K37, in DMF, 20 ° C K37, in PEG, 20. . , in air K37, in PEG, 20 ° C, in air, on silica gel K37, in PEG, 20V, in vacuum, on silica gel K37, in PEG, 37 ° C, in air K37 , in PEG, 37 ° C, in air, on silica gel K37, in PEG, 37 ° C, in vacuum, on silica gel 0 138630 139320 137241 138624 137440 138317 136942 1 week 137500 137113 137840 138801 137640 136922 136817 4 weeks 134890 134641 133980 134202 135016 135412 134097 8 weeks 135254 136021 135972 136671 135926 136201 135552 %CV 1.31 1.44 1.25 1.56 0.92 0.90 0.98 Time NR in DMF, 20 ° C NR in PEG, 20 ° C, in air NR, in PEG, 20 ° C, in air, on ruthenium NR, in PEG, 20 ° C, in vacuum, on ruthenium NR, in PEG, 37 ° C, in air NR, PEG, 37 ° C, in air, on ruthenium NR, in PEG, 37 ° C, in vacuum, on silica gel 0 495197 496130 494590 495760 494118 494241 494829 1 week 495002 495600 494802 494400 494636 493907 4946 21 4 weeks 493128 492686 493440 493662 494017 492506 492906 8 weeks 493492 494620 494525 493986 493920 492500 493650 %cv 0.21 0.31 0.12 0.19 0.06 0.19 0.18 Conclusion · (1) When reconstituted with aqueous solution, the dye incorporated into the PEG film is completely redissolved And the same fluorescence intensity as that of the dye added in the organic solvent is obtained. (2) The dye/PEG film is reproducible, and the film formed in the same manner achieves the same glory intensity® 145179.doc -50- 201109655 (3) When the most demanding storage conditions tested (37C>c, no desiccant) The dye/PEG film can remain stable for at least 52 weeks when stored under storage. Example 7 - Comparison of Amphiphilic/Nonionic Polymers • Coating of borosilicate capillaries (length 100 mm, diameter i mm, from Composite) by drawing 7 mm of a 5% polymer solution in various solvents Metal Services Ltd. - cvl 〇 12) and shake until dry. The solvent used depends on the solubility, and chloroform is favored because of its high evaporation rate. Experiments with coated capillaries were performed in triplicate and the uncoated capillaries were each implemented as a reference. The four capillaries are held vertically on the textured reference card by the two slots in the cut-in card for each capillary. The end of the hair is just 7 mm beyond the bottom of the card. The experiment was recorded using a Simkwang high-resolution, low-light color camera connected to a Winnov 500050G V10〇〇+〇V pc card, and the program Videum Capture was used to capture and analyze the data. Unless otherwise stated, the frame rate is set to 5 frames per second (to avoid losing frames). The flat bottom observation glass contains a flowing liquid (1〇_4 ^^R0se Bengal) stored in the water, and simply lowers the end of the card until the bottom of the card rests on the viewing glass wall. Immerse the capillary in the flowing liquid to a depth of 2 mm. This produces a distance of 5 mm from the bottom of the card, a distance of 9 mm from the bottom of the card to the first line, and a further 6 mm spacing from the first line up the card to the second line. The flow rate is calculated by counting the number of frames that have passed through the 9 mm stroke between the bottom of the card and the first line. 145179.doc •51 - 201109655 Table 1. Performance in terms of water column height and capillary flow rate for polymer coating/solvent systems. * - Apricot The result of a capillary, 0 = the result of two of the three capillaries, and the result is estimated when the capillary is at the height of the first line of the card month. Coating solvent average water column height mm column height STDEV j average flow rate mm/s average flow rate STDEV mm/s fastest speed mm/s PEG2000 gas pattern 17.8 0.76 21.9 3.3 25.7 PEG2000 DMF 19.8 1.89 27.6 28.1 60 PEG2000 water 13.5 5.77 29.7 37.2 72 PEG6000 gas imitation 19.3 0.76 21.5 4.8 25.7 PEG 12,000 gas imitation 16.5 0.87 7.6 3.3 9.5 PEG 20,000 chloroform 15.3 1.04 3.2 1.6 4.9 Tritonx 100 water 14.2 1.04 7.9 2.2 10 carboxymethyl cellulose water 18.0 this 180 * 180 double (2 -Aminoethyl)polyethylene glycol 2000 gas imitation 17.0 3.5 38.8 (52.5) 25.0 (10.6) 60 PEG methyl ether 5000 gas imitation 17.8 1.26 2.4 0.2 2.6 Figure 5 shows the effect of peg molecular weight on vertical flow rate. Although certain preferred embodiments of the invention have been described above and illustrated, the invention is not intended to be limited to the embodiments. Various modifications may be made thereto without departing from the scope and spirit of the invention as described in the following claims. BRIEF DESCRIPTION OF THE DRAWINGS The present invention is illustrated by the following figures, but the invention is not limited thereto. It should be understood that the figures are for illustrative purposes only. Figure 1 shows an assembly of an embodiment of a test cartridge suitable for use with a care point; Figures 2a-2c and 3a-3b show other embodiments of the assay device; Figures 4a-4c show the defined application zone, fluid flow path, detection zone and flow An example of a capillary channel that terminates the junction; and Figure 5 shows the effect of PEG molecular weight on the vertical flow rate. The error bars indicate that +/_ 1 indicates the deviation (the uncoated capillary and its first-order standard deviation are shown at 0 molecular weight). 145179.doc •52· 201109655 [Description of main components] 1 Measuring device 1 First support surface 2 Covering part 2a Bottom 2b Cover 3 Application area 3 Capillary channel 3a Well 4 Test area 4 Application area 4a Window 5 Fluid flow path 5a Flow Path 5b Flow path 5c Flow path 5d Fourth test area 6 Hydrophobic plastic strip 6 Detection area 7 Ridged handle portion 7 Flow termination knot 8 Filter 8 Alignment column 9 Hole 145179.doc -53 201109655 ίο Vent 11 pairs Quasi-hole 12 filter 13 cross-sectional area reduced fluid flow path portion 14 chamber 15 low wall or barrier 15a chamber portion 15b chamber second portion or distal region 16 raised portion 145179.doc •54·

Claims (1)

201109655 VII. Patent application scope: A measuring device for detecting the presence or quantity of an analyte present in an aqueous sample, the device comprising: at least one aqueous sample can follow a flow path of travel, human characteristics The at least one flow path comprises at least one of the two nucleophiles wherein the fluid passing along the flow path when in use is greater than would be expected by a single capillary action alone. 2. The device of claim 1, wherein the flow path is coated with the at least one amphiphilic polymer. . The device of claim 1, wherein the flow path contains the at least one amphiphilic polymer. 4. If the claim 1 is to I ^ , the device 'where the flow path is formed by the at least one amphiphilic polymer. A device of 5.^::4, wherein the flow path of the at least one amphiphilic polymer is formed by printing and/or spraying. 6_ If requested J; g j 〇 . In the apparatus of 5, wherein the at least one amphiphilic polymer is in the form of ruthenium. 7. If the at least one amphiphilic polymer is in the form of 〇, ^ ^ pellets, microspheres, nanospheres or microspheres. A device of 1, 2, or 7, wherein the at least one flow path, such as a probe, reporter, or reagent. A lesser 28 device wherein the at least one flow path contains the probe, reporter or reagent. 10. The device of claim 9, the at least one probe, the reporter molecule or the 145179.doc 201109655 11, 12 13 14 15 16 17. 18. 19. 20. 21. The reagent flows directly in the at least one Layered above or below the path, such as the device of claim 1, 2, 5 or 7, further comprising at least one application zone for applying an aqueous sample. The device of claim 11 which further comprises at least one test area in which the result and/or progress of the reaction between a portion of the aqueous sample and the at least one probe, reporter or reagent is determined. The device of claim 12 wherein the at least one flow path is in fluid communication with the at least one application region and the at least one test region. • A device as claimed in claim 1, wherein the result and/or progress of the reaction is determined by optical measurement. The device of claim 12, wherein the reaction result and/or progress is determined by visual inspection. For example, the apparatus of the long term 12 wherein the reaction result and/or progress is determined by lightening. The device of claim 16, wherein the device comprises at least two separate components. The device of claim 1 wherein the at least two separate components are readers and test cartridges. The device of claim 18, wherein the reader comprises an excitation member and a detection member. The device of claim 1, wherein the excitation member is operable to excite the test to emit fluorescence' and the detection member is operable to detect fluorescence emitted by the sample. The device of claim 18, wherein the 忒丹甲忒 test 匣 includes the at least one flow 145179.doc -2- 201109655 path. 22. 23. 24. 25. 26. 27. 28. 29. 30. The device of claim 18, wherein the test is disposable. The device of claim 18, wherein the reader is reusable. The device of claim 14, wherein the reaction is an immunoassay. The device of claim 24, wherein the reaction is ELISA. The device of claim 14, wherein the reaction is fluorescently determined. The device of claim 26, wherein the fluorescent assay is a cholesterol assay. The device of claim 26, wherein the assay is a lipoprotein assay. The device of claim 26, wherein the assay is a triglyceride assay. A test device for detecting the presence or amount of an analyte present in an aqueous sample, the device comprising: (i) at least one application zone adapted to apply an aqueous sample to the device; At least one probe, reporter molecule or reagent, wherein, at the time of use, the at least one probe, reporter molecule or reagent is capable of reacting with an analyte present in the aqueous sample; (111) at least one test area, wherein The result and/or progress of the reaction between the analyte and the at least one probe, reporter molecule or reagent can be determined in use; (iv) at least one flow path in fluid communication with the at least one application region and the at least one test region And characterized in that the at least one flow path comprises at least one amphiphilic polymer, and wherein the fluid passing along the at least one flow path in use is greater than would be expected by a single capillary action alone. 145l79.doc 201109655 31. The device of claim 30, comprising at least three test areas and at least three flow paths, wherein the first flow path is in fluid communication with the application area and the first/bay J " The second flow path is in fluid communication with the application region and the second test region, and the third flow path is in fluid communication with the application region and the third test region. 32. The device of claim 30 or 31, wherein the at least one The probe, reporter molecule or reagent is selected from the group consisting of Amplex Red, Κ37, Nile Red, cholesterol vinegarase, cholesterol oxidase or horseradish peroxidase. 33. The device of claim 32, wherein the first flow The path includes eight claws "Red", the second flow path contains K37 and the third flow path contains Nile Red 0 34. The device of claim 32, wherein the first test area comprises Red and the second test area comprises K3 7 and The three test areas contain Nile Red. 35. The device of claim 34, wherein the first flow path further comprises cholesterol esterase, cholesterol oxidase, and horseradish peroxidase. 36. The device of claim 34 wherein the first test region further comprises cholesterol esterase, cholesterol oxidase, and horseradish peroxidase. 37. The device of claim 32, wherein the application region comprises a probe, report of the at least one selected from the group consisting of Amplex Red, Κ37, Nile Red, cholesterol vinegarase, cholesterol oxidase or horseradish peroxidase Molecule or reagent. 38. The device of claim 30 or 31, wherein the at least one probe, reporter molecule or reagent is in dry form. 145I79.doc 201109655 39. 40. 41. 42. 43. 44. 45. 46. 47. The device of claim 38, wherein the at least one probe, reporter molecule or reagent comprises a stabilizer. Such as. The device of item 39, wherein the stabilizer is. A method for measuring lipoprotein in an aqueous biological sample, comprising: (1) contacting the aqueous biological sample with a combination of at least one hydrophobic luminophore and at least one amphiphilic polymer, wherein the at least one hydrophobicity The luminophore binds to at least one lipoprotein in the aqueous biological sample and, when bound thereto, fluoresces under appropriate excitation; () excites from step (i) at an excitation wavelength between about 40 〇 nm and 520 nm Product; ((1)) Measure the fluorescence emission after step (u) at a wavelength between about 490 nm and 650 nm. A method of claim 41, wherein the aqueous biological sample is contacted with the at least one amphiphilic polymer and then contacted with at least one hydrophobic luminophore. The method of claim 4, wherein the contacting of the aqueous biological sample with the at least one amphiphilic polymer is substantially simultaneous with its contact with the at least one hydrophobic luminophore. A method of enhancing lateral fluid flow comprising applying an amphiphilic polymer to a fluid along a surface of the flow. The method of claim 44, wherein the surface is defined by a tube, capillary, channel, well or membrane. The use of a Gafquat is used to stabilize the enzyme. Use of a device according to any one of claims 1 to 4 in the assay. 145179.doc