Glycated hemoglobin measurement

Abstract

This article describes the equipment, systems, and methods used to measure glycated hemoglobin.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 040,159, filed June 17, 2020, and U.S. Provisional Patent Application No. 62 / 877,188, filed July 22, 2019, the entire disclosure of each of which is incorporated herein by reference. Technical Field

[0003] This article describes the equipment, systems, and methods used to measure glycated hemoglobin. Summary of the Invention

[0004] This article provides an overview of the equipment, systems, and methods used for hemoglobin measurement, particularly glycated hemoglobin (HbA1c). Determining the level of HbA1c in a patient sample is a key component of the diagnosis of type 1 and type 2 diabetes, as well as gestational diabetes, because it allows for the estimation of an individual's average blood glucose level over a period of time (e.g., three months).

[0005] In some implementations, this glycated hemoglobin measurement can be a whole blood measurement. Whole blood can be human or animal whole blood. However, various components of the blood can also be measured as long as hemoglobin components are present.

[0006] Typically, measurements can be performed using microscopic slide test elements or simply microscopic slides such as dry slide test elements. These microscopic slides are used in automated analyzers. The microscopic slide can be a single slide, allowing the entire analysis to be completed using one or more drops of sample and a single slide instead of multiple slides. In some implementations, multiple measurements can be performed on a single slide.

[0007] The microscope slides described herein may include a stack of films, the stack comprising, from bottom to top, a first film layer, a second film layer, and a third film layer. The first film layer comprises a cross-linked gel, wherein the cross-linked gel comprises a detection agent, a fructose oxidase, and a peroxidase. The second film layer comprises the first gel, and the third film layer comprises a lysing agent, a denaturing agent, and a protease. In some embodiments, the first film layer is a gel layer, the second film layer is a masking layer, and the third film layer is a spreading layer. In some embodiments, the microscope slide may include an adhesion layer or sublayer between the masking layer and the spreading layer.

[0008] The microscope slides described herein may include a stack of films, the stack comprising, from bottom to top, a first film layer, a second film layer, and a third film layer. The first film layer comprises a cross-linked gel, wherein the cross-linked gel comprises a detection agent, a fructose oxidase, an anti-interference agent, and a peroxidase. The second film layer comprises the first gel, and the third film layer comprises a lysing agent, a denaturing agent, and a protease. In some embodiments, the first film layer is a gel layer, the second film layer is a masking layer, and the third film layer is a spreading layer. In some embodiments, the microscope slide may include an adhesion layer or sublayer between the masking layer and the spreading layer.

[0009] The microscope slides described herein may include a stack of film layers, the stack comprising, from bottom to top, a gel layer, a masking layer, and a spreading layer. In other embodiments, the microscope slides described herein may include a stack of film layers, the stack comprising, from bottom to top, a gel layer, a masking layer, an adhesive layer or sublayer, and a spreading layer. In some embodiments, the first layer or gel layer comprises a gel. In some embodiments, the second film layer, i.e., the masking layer, comprises a second gel. In some embodiments, the third film layer, i.e., the spreading layer, comprises a lysing agent, a denaturing agent, and a protease. In some embodiments, an adhesive layer or sublayer is included as the third layer, and the spreading layer is the fourth layer.

[0010] In some embodiments, the gel may be a cross-linked gel. Other layers may be included in the microscope slide. In some embodiments, the cross-linked gel contains a detection agent, fructose oxidase, and peroxidase.

[0011] The first membrane layer may also contain an oxidase cofactor and a surfactant. In some embodiments, the oxidase cofactor is flavin adenine dinucleotide (FAD). Fructose-based oxidase may be specific for Fru-α-Val His peptide or glycosylated amino acids such as Fru-α-Val. In some embodiments, the anti-interference agent is ascorbic acid oxidase (AAO).

[0012] In some embodiments, the detection reagent is a leuco dye, such as a blue leuco dye. The detection reagent may be selected from sodium N-carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine (DA-64), N,N,N'N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triamino-triphenylmethane hexasodium salt (TPM-PS), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenothiazine sodium (DA-67), and 2-(3,5-dimethoxy-4-hydroxyphenol)-4,5-bis-(4-dimethylaminophenyl)imidazole.

[0013] In some implementations, the peroxidase is horseradish peroxidase.

[0014] In some embodiments, the second film layer further comprises a reflective material portion. The reflective material portion may comprise a metal salt. In one embodiment, the metal is titanium, such as, but not limited to, titanium dioxide (TiO2).

[0015] In some implementations, the third membrane layer also contains calcium.

[0016] In some embodiments, the third membrane layer comprises a porous layer containing latex particles. In some embodiments, the latex particles may be formed from a vinyltoluene-co-methacrylic acid copolymer (VtE). The particles, or sometimes referred to as beads, may have a median particle size of about 25 µm. In one embodiment, the particles may have a median particle size of less than 25 µm. In other embodiments, the particles may have a median particle size of about 10 µm to about 40 µm, about 15 µm to about 35 µm, about 20 µm to about 30 µm, less than about 15 µm, less than about 10 µm, or less than about 5 µm.

[0017] In some embodiments, the pyrolysis agent is a detergent. The detergent may be selected from octylphenol ethoxylate (TRITON® X-100, Union Carbide Corporation (New York)), TWEEN® (ICI Americas Inc. (Delaware)) (TWEEN 20), sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ether (POE), and NONIDET® (Air Products and Chemicals, Inc. (Delaware)) P-40 (NP-40). In one embodiment, the detergent is TRITON X-100.

[0018] In some embodiments, the denaturant is an oxidant or a surfactant. The denaturant may be one or more of sodium nitrite or N-lauroyl sarcosine (NLS).

[0019] In some embodiments, the protease is a metalloproteinase and / or a neutral protease. The protease may be an endopeptide or an exopeptide. In other embodiments, the protease is selected from proteinase K, streptoprotein E, proteinase XVII, proteinase XXI, aminopeptidase, carboxypeptidase, thermophilic protease, bacillolysin (a microbial metalloproteinase), peptidase K, endopeptidase K, chymotrypsin, chymotrypsin C, gamma-glutamyl endopeptidase, peptidyl-lys-metalloendopeptidase, proteases from Bacillus, leucine aminopeptidase, and subtilisin.

[0020] In some embodiments, the third film layer is in direct contact with the upper surface of the second film layer. In other embodiments, the second film layer is in direct contact with the upper surface of the first film layer.

[0021] In some implementations, the spread layer is in direct contact with the upper surface of the masking layer. In other implementations, the spread layer is in direct contact with the upper surface of the adhesive layer.

[0022] This document also describes single-slide methods for detecting hemoglobin and glycated hemoglobin. These methods include a) providing a microscope slide as described herein; b) contacting a third membrane layer of the microscope slide with an untreated blood sample containing red blood cells, wherein a lysing agent releases glycated hemoglobin from the red blood cells, wherein a denaturing agent contacts the glycated hemoglobin to denature it, and wherein a protease releases fructosyl peptides from the denatured glycated hemoglobin, wherein the fructosyl peptides reach a first membrane layer and react with fructosyl oxidase and FAD cofactor to generate peroxides, and wherein the peroxidase and peroxides react with a detection agent to release a detectable signal; c) measuring the amount of hemoglobin from the blood sample, wherein measuring the amount of hemoglobin includes measuring the sample reflectance density from said slide under light of a first wavelength; and d) measuring the amount of glycated hemoglobin from the blood sample, wherein measuring the amount of glycated hemoglobin includes measuring the reflectance density from the detection agent under light of a second wavelength, wherein the second wavelength of light is different from the first wavelength of light. In some embodiments, the detection agent is an oxidizing dye.

[0023] In some embodiments, the untreated blood sample may be an unlyzed blood sample. In some embodiments, the untreated blood sample may be blood that has been treated with an anticoagulant but not with a lysing agent. In some embodiments, the anticoagulant is an anticoagulant. In some embodiments, the untreated blood may be whole blood.

[0024] In some implementations, the first wavelength of light is 540 nm and the second wavelength of light is 670 nm.

[0025] In some implementations, the method also includes contacting the fructose-based peptide with an oxidase cofactor such as flavin adenine dinucleotide (FAD).

[0026] This article also describes single-slide methods for the direct detection of glycated hemoglobin. These methods include a) providing a microscope slide in which a second membrane layer comprises a portion of reflective material; b) contacting a third membrane layer of the slide with a blood sample containing untreated red blood cells, wherein a lysing agent releases glycated hemoglobin from the red blood cells, wherein a denaturing agent contacts the glycated hemoglobin to denature it, and wherein a protease releases fructosyl peptides from the denatured glycated hemoglobin, wherein the fructosyl peptides cross the second membrane layer, wherein the fructosyl peptides reach the first membrane layer and react with fructosyl oxidase and FAD cofactor to generate peroxides, and wherein the peroxidase and peroxides react with a detection agent to release a detectable signal; and c) measuring the amount of glycated hemoglobin from the blood sample, wherein measuring the amount of glycated hemoglobin includes measuring the reflectance density of an oxidized dye in the blood sample.

[0027] In some implementations, the reflective material contains a metal salt.

[0028] Due to the porosity created by latex particles in the third membrane layer, whole blood samples can pass through this layer. Attached Figure Description

[0029] Figure 1 The diagram illustrates the layers incorporated into a microscope slide as described herein.

[0030] Figure 2 The illustration shows a comparison between a glass slide with TiO2 in its masking layer and a glass slide without TiO2 in its masking layer.

[0031] Figure 3A and 3B The diagram illustrates the 540 nm signal of a masking layer without TiO2 (“control”) and a masking layer containing TiO2.

[0032] Figure 4A and 4B The diagram illustrates the 670 nm signal of a masking layer without TiO2 (“control”) and a masking layer containing TiO2.

[0033] Figure 5 The dose response of the Fru-VH substrate is illustrated for a glass slide in which TiO2 is not present in the presence of increased hemoglobin (Hb) concentration.

[0034] Figure 6 The dose response of the Fru-VH substrate is illustrated for a glass slide containing TiO2 in its masking layer in the presence of increased hemoglobin (Hb) concentration.

[0035] Figures 7A-7DThe diagram illustrates the kinetic data of the %A1c model fluid and the %A1c patient sample at 670 nm over a 5-minute time period on an analyzer at 37°C. Protease and sodium nitrite were deposited on the finished microscopic slides by inkjet printing and then dried.

[0036] Figure 8A and 8B The diagram illustrates the kinetic data of the %A1c model fluid and the %A1c patient sample at 670 nm over a 5-minute time period on an analyzer at 37°C. All components were incorporated via an X-hopper coating process.

[0037] Figure 9 This illustrates the reflectance density dose-response data of the %A1c model fluid and the %A1c patient sample at 670 nm over a 5-minute time period on an analyzer at 37°C. All components were incorporated via an X-hopper coating process.

[0038] Figure 10 The correlation diagram between %A1c of the microscopic slide and %A1c of BioRad Variant HPLC is shown.

[0039] Figure 11 The dose response of patient samples of hemoglobin components on a microscopic slide is illustrated.

[0040] Figure 12 The diagram illustrates the correlation between the determination of hemoglobin components using a microscopic slide and the determination of hemoglobin components using a MicroTip reference.

[0041] Figure 13 The dose response of HbA1c fraction patient samples on a microscopic slide is illustrated.

[0042] Figure 14 The diagram illustrates the correlation between HbA1c composition determination on a microscopic slide and HbA1c composition determination on a MicroTip reference slide.

[0043] Figure 15 The diagram illustrates the correlation between the %A1c determination derived from the microscope slide and the %A1c determination referenced by HPLC.

[0044] Figure 16 The diagram illustrates the HbA1c enzymatic dose response at 670 nm for a dual-measurement microscope slide test element.

[0045] Figure 17 The hemoglobin spectrum at 540 nm dose response of the dual-measurement microscopy slide test element is illustrated. Detailed Implementation

[0046] This describes the use of patient samples to measure glycated hemoglobin concentration via an enzymatic cascade using a thin-film testing element—a microscopic slide—either by direct means (%A1c measurement only) or by deductive calculation (HbA1c and hemoglobin measurements to derive %A1c results). In some embodiments, the patient sample is a whole blood sample, such as an untreated whole blood sample. In some embodiments, the untreated blood sample may be an unlysaturated blood sample. In some embodiments, the untreated blood sample may be blood that has been treated with an anticoagulant but not with a lysis agent. In some embodiments, the anticoagulant is an anticoagulant. In some embodiments, the untreated blood may be whole blood.

[0047] The blood sample can be human or animal blood. In some implementations, the blood can be mammalian blood. Mammals can include, but are not limited to, humans, horses, camels, dogs, cats, cattle, bears, rodents, sheep, goats, pigs, etc. Blood from other animals such as reptiles, fish, and birds can also be used.

[0048] In some implementation schemes, various components or portions of a patient's blood sample can be measured as long as hemoglobin components are present.

[0049] The measurements were performed using the equipment, systems, and methods described herein. Typically, measurements can be performed using microscope slides, such as dry microscope slides. The microscope slide can be a single slide, allowing the entire analysis to be completed using a single drop of sample and a single slide instead of multiple slides.

[0050] The microscope slides described herein can be used in automated analyzer systems or other types of mainframe analyzers. In some implementations, these types of instruments can process hundreds or even thousands of sample analyses per workday. In one implementation, the microscope slides can be used in current VITROS mainframe analyzers (5,1 FS, 4600 Chemical System, 5600 Integrated System) as well as future VITROS analyzers. Additionally, the microscope slides described herein can be used in systems manufactured by Abbott Laboratories, Beckman Coulter, Baxter, Genprobe, Roche Diagnostics, and Siemens.

[0051] However, in other implementations, microscope slides may be used in non-automated or semi-automated systems. In some implementations, microscope slides may be used on a sample-by-sample basis and / or loaded manually.

[0052] In some implementations, the microscopy slide testing element described herein can incorporate components of an enzymatic cascade. This enzymatic cascade can result in the generation of a colorimetric signal directly correlated with the concentration of glycated hemoglobin (%A1c) in a patient sample.

[0053] In the industry, hemoglobin glycation levels are typically determined by measuring both hemoglobin (Hb) and glycated hemoglobin (HbA1c) concentrations and expressing them as a ratio (derived %A1c). This requires two sets of calibrators to generate separate calibration curves for determining Hb and HbA1c. Alternatively, a fluid with a known %A1c can be used as a calibrator to calibrate the assay, providing the %A1c directly for unknown patient samples.

[0054] The apparatus, systems, and methods described herein can utilize microscope slides suitable for any measurement format. In one embodiment, all components required for determining Hb and HbA1c concentrations can be incorporated into a single test element to obtain a derived %A1c result. In such an embodiment, Hb and HbA1c can be measured from a single slide at different detection wavelengths, the test can be performed from a single whole blood measurement event, and current microscope slide protocols can be used.

[0055] In another implementation, a second option is to use separate microscope slides to measure Hb and HbA1c concentrations separately to obtain the derived %A1c result. In such an implementation, Hb and HbA1c concentrations can be determined from separate test slides in a single test element at different detection wavelengths, the test can be performed from two whole blood measurement events, and can be performed using current microscope slide protocols.

[0056] Furthermore, in another embodiment, %A1c can be measured directly using a single microscope slide. In such an embodiment, %A1c can be determined from a single microscope slide at a single detection wavelength. This measurement can be performed from a single whole blood measurement event and can be performed using current microscope slide protocols.

[0057] In some embodiments, the apparatus, system, and method may use an enzyme cascade to determine HbA1c in the form of %A1c. The enzyme cascade used in the microscope slides described herein is as follows:

[0058]

[0059] In some implementations, this cascade can be used to directly measure glycated hemoglobin (as %A1c). However, the cascade can also be used when measuring derived %A1c.

[0060] Typically, methods for determining %A1c, either directly or indirectly, involve applying an untreated whole blood sample to a microscope slide as described herein. In some embodiments, the microscope slide may include two or more sample sites and may require more than one blood sample.

[0061] A cleavage surfactant cleaves red blood cells in a blood sample, releasing glycated hemoglobin. A second surfactant denatures the glycated hemoglobin, providing a pathway to the proteolytic cleavage site. A protease then cleaves the N-terminal portion of the hemoglobin β-chain, releasing a glycated dipeptide (fructosyl-α-valine-histidine-Fru-α-ValHis). The glycated dipeptide is then deglycated in an enzymatic reaction using fructosyl peptide oxidase (FPOX) and flavin adenine dinucleotide (FAD), producing hydrogen peroxide (H2O2). H2O2 and horseradish peroxidase (HRP) oxidize a leuco dye, generating a colorimetric signal at 670 nm. The concentration of glycated hemoglobin is proportional to the reflectance density of the resulting dye. In some embodiments, the hemoglobin signal can be read at 540 nm, and the derived %A1c value can be determined using the hemoglobin component measurements at 540 nm and the glycated hemoglobin measurements at 670 nm.

[0062] In some embodiments, the microscope slide may include at least a first film layer, a second film layer, and a third film layer. The microscope slide may contain more layers. In some embodiments, the first film layer may comprise cross-linked gelatin or a gel, wherein the cross-linked gel comprises a detection agent, a fructose oxidase, a peroxidase, and optionally an anti-interference agent. In some embodiments, the second film layer may comprise gelatin, a gel, or a cross-linked gel or gelatin. In some embodiments, the third film layer may comprise a lysing agent, a denaturing agent, and / or a protease.

[0063] The microscope slide 100 may comprise a stack of film layers or simply a stack of layers. For example... Figure 1 As illustrated, the stacking of film layers may include a gel layer 102, a masking layer 104, an adhesion layer 106, and a spreading layer 108. In some embodiments, the microscope slide 100 may include an upper slide mount 110, a lower slide mount 112, or both. In some embodiments, these layers may be constructed on a support layer 114 during formation. In some embodiments, the adhesion layer 106, the masking layer 104, and the gel layer 102 may be combined into a reagent layer.

[0064] In some embodiments, the support layer 114 may be formed of polyethylene terephthalate or other suitable transparent polymer materials. This transparent polymer material allows for the application or coating of layers thereon.

[0065] In some embodiments, the upper slide mount 110 and the lower slide mount 112 are formed of polystyrene or other suitable polymer materials.

[0066] Each layer and mounting element can be combined to form a microscope slide with an upper surface 116 and a lower surface 118 that are square or generally rectangular. In some embodiments, the upper surface 116 and the lower surface 118 may have other shapes, such as, but not limited to, triangles, pentagons, hexagons, heptagons, octagons, circles, ovals, ellipses, or other shapes composed of straight lines or circles.

[0067] In some embodiments, the microscope slide may include at least one groove or keying surface. The groove or keying surface can be used to facilitate the stacking of multiple microscope slides and / or the loading of one or more microscope slides into an analytical instrument. In one embodiment, microscope slide 100 may include a groove 120. The groove 120 is shown as having a shape composed of straight lines, but in other embodiments, the groove 120 may actually be any shape that allows stacking and / or loading.

[0068] Furthermore, the microscope slide 100 may include a window portion 122 on the upper surface 116 and / or the lower surface 118. The window portion 122 may be surrounded by a frame portion 124. However, in some embodiments, the frame portion is not included, and the window portion 122 may extend to the edge of the microscope slide.

[0069] The microscope slide 100 may also include a sample area 126 within a window portion 122 on the upper surface 116. The sample area 126 can serve as the location for applying a sample. On the lower surface 118, a detection area 128 may exist within the window portion 122. The detection area 128 can serve as the location for detection using an analyzer.

[0070] Here, these layers will be described as they pass through the microscope slide as the sample travels. Spread layer 108 may be the first layer to which the sample interacts. Spread layer 108 may contain polymer beads, binder, buffer, at least one surfactant, divalent cationic salt, sodium nitrite, protease, alcohol, and water.

[0071] In some implementations, the divalent cation salt may be calcium chloride or any compound that can bind to EDTA.

[0072] In some implementations, tert-butanol may be present during the formation or application of the spread layer. However, tert-butanol is not present after drying.

[0073] In some embodiments, the polymer beads may include acrylic beads, such as, but not limited to, vinyltoluene-co-methacrylic acid copolymer beads (VtE beads). The beads may function to create pores in the spread layer that allow red blood cells to enter the coating. The beads may also provide a white reflective surface, promote uniform sample spread, and trap interfering substances such as heme byproducts, catalase, triglycerides, etc.

[0074] The beads may have a sufficiently large average diameter to allow red blood cells to penetrate the coating. In some embodiments, the diameter is greater than about 5µm, greater than about 10µm, greater than about 50µm, greater than about 80µm, between about 20µm and about 100µm, between about 20µm and about 30µm, between about 10µm and about 40µm, between about 10µm and about 100µm, between about 20µm and about 25µm, between about 50µm and about 100µm, between about 25µm and about 30µm, less than about 100µm, less than about 80µm, less than about 50µm, less than about 40µm, or less than about 30µm.

[0075] The pores created by the beads can have pore sizes greater than about 5µm, greater than about 10µm, greater than about 20µm, between about 20µm and about 30µm, between about 10µm and about 40µm, between about 20µm and about 25µm, between about 25µm and about 30µm, less than about 50µm, less than about 40µm, or less than about 30µm. In one embodiment, the pore size is about 25µm.

[0076] The binder that can be used to promote interlayer cohesion may be a latex. In one embodiment, the monomer molecular weight (MWM) of the latex in the final product may be about 30% of the latex solids. In some embodiments, the latex contains a biocidal agent such as, but not limited to, nipacide. In other embodiments, alternative binders such as, but not limited to, polyacrylamide (I100) may be used.

[0077] A buffer is used to maintain the layer at a desired pH. The desired pH may be about 6.0 to about 7.0, about 6.2 to about 7.2, about 6.5 to about 7.5, about 6.0 to about 8.0, about 7.0 to about 8.0, about 6.8 to about 7.2, about 7.4, about 7.2, about 7.0, or about 6.8. The buffer may be an acid or a base, as needed. In one embodiment, the buffer is 3-(N-morpholino)propanesulfonic acid (MOPS). Other buffers may include, but are not limited to, sodium bicarbonate, calcium carbonate, potassium phosphate, tris(hydroxymethyl)aminomethane (TRIS), Bicine, Bis-TRIS, TES, HEPPS (EPPS), and combinations thereof.

[0078] Some implementations may include a first surfactant and a second surfactant. The first surfactant may be a lysing agent. The lysing agent lyses red blood cells and releases hemoglobin and glycated hemoglobin. The lysing agent may be a detergent.

[0079] In some embodiments, the detergent may be selected from octylphenol ethoxylate (TRITON X-100), TWEEN (TWEEN 20), sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ether (POE), NONIDET P-40 (NP-40), or combinations thereof. In one embodiment, the detergent is an octylphenol ethoxylate such as TRITON X-100.

[0080] In some embodiments, the second surfactant can be a denaturant that denatures hemoglobin. This denaturant exposes sites on the hemoglobin for protein hydrolysis. The denaturant can oxidize heme to a single oxidation state and facilitate the conversion of hemoglobin forms (oxygenated, deoxygenated, carboxylated) to a single spectral form.

[0081] In one embodiment, the denaturing agent may include N-lauroyl sarcosine (NLS). In some embodiments, the second surfactant may also include a denaturing aid or an oxidizing agent such as sodium nitrite. The denaturing aid may help promote denaturation by NLS by coordinating with iron in heme.

[0082] In one embodiment, a denaturing agent (one or more) present in the coating denatures glycated hemoglobin, thereby providing access to the target protease cleavage site.

[0083] In some implementations, the spreading layer 108 may contain other surfactants as needed to initiate an enzymatic cascade.

[0084] Sodium nitrite can exist in molar excess. In some embodiments, sodium nitrite (NaNO2) can be present at approximately 5-10 times the total hemoglobin concentration. In some embodiments, sodium nitrite can act to oxidize heme to the ferric (+3) state.

[0085] Furthermore, the combination of denatured surfactant, sodium nitrite, and hemoglobin can produce a single spectral form of hemoglobin that can be read at 540 nm.

[0086] The protease can be a neutral protease. The protease can be a metalloproteinase. The protease can be an endopeptide or an exopeptide. The protease can generate Fru-α-ValHis by cleavage from the N-terminus of the hemoglobin β subunit or chain. Fru-α-ValHis can be a substrate of the fructose oxidase contained in the gel layer described herein.

[0087] In some embodiments, the protease may be proteinase K, streptinase E, protease XVII, protease XXI, aminopeptidase, carboxypeptidase, thermophilic protease, subtilisin, or a combination thereof.

[0088] Fru-α-ValHis dipeptide has a sufficiently small molecular weight so that it can easily pass through the adhesion layer and masking layer and enter the gel layer described herein.

[0089] Calcium may be essential for protease activity. In some embodiments, calcium in the microscope slide protects the protease from the EDTA anticoagulant in the blood collection tube. In some embodiments, the calcium source is calcium chloride (CaCl2). In other embodiments, the calcium chloride is calcium chloride dihydrate.

[0090] Calcium chloride present in the spread layer can protect protease activity. In some clinical settings where HbA1c is measured, blood collection tubes are EDTA plasma tubes. In the absence of calcium chloride, EDTA may bind to calcium and zinc from proteases, significantly reducing their proteolytic activity.

[0091] The solvent for the spreading layer may be methanol, ethanol, tert-butanol, or combinations thereof. In one embodiment, the alcohol is approximately 97% by weight tert-butanol.

[0092] Adhesion layer 106 is located immediately below spread layer 108 and may contain an adhesive substance, surfactant, and / or solvent. The Fru-α-ValHis dipeptide generated in the spread layer can easily penetrate adhesion layer 106.

[0093] In some embodiments, the adhesive material can serve to promote adhesion between the spread layer 108 and the masking layer 104. In one embodiment, the adhesive material is polyisopropylacrylamide (l100). In other embodiments, the adhesive material may be polyvinylpyrrolidone (PVP). In some embodiments, PVP may have a k90 chain length, a k30 chain length, a k15 chain length, or a combination thereof.

[0094] The adhesive layer surfactant can act as a coating aid. In some embodiments, the adhesive layer surfactant is an octylphenol ethoxylate such as TRITON X-100. In some embodiments, the adhesive substance may be a combination of polyisopropylacrylamide and an octylphenol ethoxylate.

[0095] The solvent in the adhesive layer 106 may be ethanol, isopropanol, methanol, tert-butanol, acetone, or a combination thereof. In one embodiment, the solvent for the adhesive layer may be acetone. In some embodiments, when using ethanol, a surfactant may not be required.

[0096] The masking layer 104 may comprise a gel, at least one buffer, pigment substance, dispersant, surfactant, hardener, and / or solvent / diluent. In some embodiments, the gel is gelatin and / or a hardened gel.

[0097] The gel can promote layer cohesion, enhance capillary forces during rewetting, and / or provide size exclusion mechanisms. In some embodiments, the size exclusion mechanism can exclude high molecular weight interfering substances (during crosslinking / curing).

[0098] In some embodiments, the gel is a hardened gel. In one embodiment, the gel is Gel-RCRousselot Dub Pig Dia Type 56 or 275 Bloom Type A NF pigskin gelatin.

[0099] In some embodiments, the pigment material can create reflective portions within the masking layer. The pigment material provides a white reflective surface and acts to capture or mask interfering substances such as heme byproducts, catalase, and triglycerides. In some embodiments, the pigment material may include a metallic substance or metal. In some embodiments, the metal is titanium, such as, but not limited to, titanium dioxide (TiO2). Titanium dioxide can be anatase titanium dioxide pigment with high whiteness and a blue tint. In some embodiments, the pigment material is Hombitan LC-S, Huntsman TiO2, or Kemiera 300. In other embodiments, titanium dioxide may be in other crystalline forms such as, but not limited to, rutile, brookite, akaogiite, and combinations thereof, or combinations with anatase.

[0100] In some embodiments, titanium dioxide and a hardened gel can work together to create a sieve, in which small molecular weight species (such as Fru-α-ValHis) can easily pass through the layer, while larger molecular weight proteins (hemoglobin, catalase, protease) are excluded. In one embodiment, a sieve that allows Fru-α-ValHis to pass through is created.

[0101] In some implementations, this exclusion of larger molecular weight proteins can be a fundamental feature of the masking layer, since hemoglobin can cause optical interference in HbA1c measurements at 670 nm, catalase can consume the peroxides required for dye oxidation, and / or proteases can digest the registration enzymes (fructosyl peptidase, horseradish peroxidase) present in the gel layer.

[0102] In some embodiments, titanium dioxide can provide a uniform reflective surface for reflecting light from an analyzer light source to a detector for signal quantification. The analyzer light source can be a light-emitting diode (LED) or other light source capable of providing light at the wavelengths described herein. After light contacts or otherwise interacts with the sample, sensors (one or more) can be used to read the amount of light at one or more wavelengths. Sensors can be photomultiplier tubes, contact image sensors, image capture sensor matrices, or combinations thereof.

[0103] The buffered masking layer can be used to maintain the masking layer at a desired pH. The desired pH may be about 6.0 to about 7.0, about 6.2 to about 7.2, about 6.5 to about 7.5, about 6.0 to about 8.0, about 7.0 to about 8.0, about 6.8 to about 7.2, about 7.4, about 7.2, about 7.0 or about 6.8.

[0104] In one embodiment, at least one masking layer buffer may comprise a first buffer and a second buffer, wherein each buffer may be an acid salt or a base salt as needed. In one embodiment, the first buffer is 3-(N-morpholino)propanesulfonic acid (MOPS). In one embodiment, the second buffer is disodium β,β-dihydroxy-1,4-piperazine dipropanesulfonate (POPSO).

[0105] The dispersant may be sodium polymethacrylate. This agent is effective for rapidly dispersing pigments. In one embodiment, the dispersant is Daxad 30S.

[0106] The masking surfactant can act as a coating aid. The masking surfactant can be anionic surfactants such as polyether sulfonates. In one embodiment, the masking surfactant is TRITON X200E.

[0107] A hardener can be used to gel and / or promote cohesion within the crosslinked gel layer. In some embodiments, the hardener is bis(vinylsulfonylmethyl) (BVSM).

[0108] In some implementations, the masking layer solvent / diluent is water.

[0109] The masking layer 104 can separate the functional areas of the slide. For example, the masking layer 104 can separate the spreading layer 108 from the gel layer 102. This can separate the red blood cell lysis, hemoglobin denaturation / digestion, and Fru-α-ValHis dipeptide release that occur in the spreading layer from the Fru-α-ValHis dipeptide desaccharification and HRP / dye reaction that occur in the gel layer 102 to generate a colorimetric signal.

[0110] In some implementations, no masking layer is present. For example, when forming a slide that requires Hb measurement at 540 nm, titanium dioxide is not used because it would hinder the ability to read hemoglobin.

[0111] However, in other embodiments where Hb needs to be measured at 540 nm, the masking layer 104 may still comprise titanium dioxide. In some embodiments, a reflected signal of Fru-α-ValHis at 670 nm may be generated within the gel layer, and a reflected signal of Hb not penetrating the masking layer at 540 nm may be generated above the masking layer. In such embodiments, two separate reflected signals are measured, one for Hb measured from above at 540 nm and one for Fru-α-ValHis measured from below at 670 nm. These values ​​can be used to determine the derived %HbA1c.

[0112] The gel layer 102 may contain a gel, a buffer, at least one surfactant, a colorant solvent, a reducing agent, a detection agent, a cofactor, an amplifying substance or catalyst, an oxidase, a curing agent, and a solvent / diluent.

[0113] In some embodiments, the gel layer is a gel or gelatin. The gel can promote layer cohesion, enhance capillary forces during rewetting, and / or provide a size exclusion mechanism. In some embodiments, the size exclusion mechanism can exclude high molecular weight interfering substances (during crosslinking / curing). In some embodiments, the gel is a crosslinked gel. Crosslinking can improve layer integrity and reduce pore size to filter out additional interfering substances.

[0114] In one embodiment, the gel is Gel-32 TCGIII DI gelatin. The gel can be porous.

[0115] In some implementations, the gel can act as a size exclusion mechanism.

[0116] A gel layer buffer can be used to maintain the layer at a desired pH. The desired pH can be about 6.0 to about 7.0, about 6.2 to about 7.2, about 6.5 to about 7.5, about 6.0 to about 8.0, about 7.0 to about 8.0, about 6.8 to about 7.2, about 7.4, about 7.2, about 7.0, or about 6.8. The buffer can be an acid or a base, as needed. In one embodiment, the buffer is 3-(N-morpholino)propanesulfonic acid (MOPS).

[0117] The gel layer 102 may contain one or more surfactants. The first surfactant may act as a coating aid. In some embodiments, the first surfactant of the gel layer is an octylphenol ethoxylate such as TRITON X-100.

[0118] The second surfactant can be used to disperse the dye. In one embodiment, the second surfactant is alkylated sodium naphthalenesulfonate, such as Alkanol XC.

[0119] The colorant solvent for the dye layer can be 2,4-di-n-pentylphenol (KS-52) and / or 2,4-di-tert-pentylphenol (KS-41).

[0120] A reducing agent may be added to prevent the oxidation of the false dye. In one embodiment, the reducing agent may be 5,5-dimethyl-1,3-cyclohexanedione (Dimedone).

[0121] The detection reagent may be a dye. The detection reagent can be used to generate a colorimetric signal. Almost any dye that provides a detectable signal can be used. The dye may be sodium N-carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine (DA-64), N,N,N'N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triamino-triphenylmethane hexasodium salt (TPM-PS), sodium 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenothiazine (DA-67), 2-(3,5-dimethoxy-4-hydroxyphenol)-4,5-bis-(4-dimethylaminophenyl)imidazolium, or a combination thereof. In one embodiment, the dye is 2-(3,5-dimethoxy-4-hydroxyphenol)-4,5-bis(4-dimethylaminophenyl)imidazolium.

[0122] The oxidase may be an oxidase that produces peroxides from Fru-α-ValHis. The oxidase can desaccharify Fru-α-ValHis to produce peroxides. In one embodiment, the oxidase may be a fructosyl peptide oxidase.

[0123] In some embodiments, gel layer 102 may contain a cofactor for oxidase reactions. The cofactor may be a non-protein compound that contributes to oxidase activity. In other embodiments, the cofactor may be flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and / or coenzyme A (CoA). In one embodiment, the cofactor may be flavin adenine dinucleotide (FAD).

[0124] The amplifying agent or catalyst can be any molecule that amplifies the reaction of a colorimetric dye. Peroxides produced by the Fru-α-ValHis oxidase reaction can interact with the dye, and their signal is amplified by the amplifying agent. In some embodiments, the amplifying agent is a peroxidase, such as, but not limited to, horseradish peroxidase (POD).

[0125] A hardener can be used to gel and / or promote cohesion within the crosslinked gel layer. In some embodiments, the hardener is bis(vinylsulfonylmethyl) (BVSM).

[0126] In some implementations, the solvent / diluent for the gel layer is water.

[0127] In some embodiments, Fru-α-ValHis is desaccharified by fructosyl peptide oxidase in gel layer 102. Fructosyl oxidase is specific for both Fru-α-ValHis and Fru-α-Val. However, Fru-α-Val is not produced by the proteolytic hydrolysis of the hemoglobin β-chain. The specificity of fructosyl oxidase also prevents interference from other glycated proteins such as albumin in the assay.

[0128] Desaccharification results in the production of peroxides via a FAD cofactor cycle. Horseradish peroxidase and peroxides oxidize the dye into a colored product that absorbs light at 670 nm.

[0129] In some embodiments, the hardened gel in gel layer 102 serves as an additional protective layer to exclude larger molecular weight proteins (hemoglobin, catalase, protease). The cross-linking of the gel reduces the pore size of this layer and also helps prevent dye particles from the gel layer from mixing with masking layer (e.g., melt) components during coating.

[0130] In some embodiments, hardening of the gel layer 102 can increase the signal of the microscope slide. Hardening can increase the signal by about 5% to about 10%, about 1% to about 10%, about 1% to about 5%, about 5% to about 20%, or about 1% to about 20%.

[0131] In some embodiments, the anti-interference agent may be ascorbic acid oxidase. This oxidase reacts with ascorbic acid (vitamin C) in the sample to prevent ascorbic acid from interfering with the measurement. In some embodiments, ascorbic acid in the sample may react with dyes in the gel layer to reduce them and weaken their color. This decolorization may lead to negative %A1c prediction bias, or simply result in falsely low %A1c results. By including ascorbic acid oxidase, ascorbic acid in the sample can be eliminated, thereby preventing negative prediction bias.

[0132] In some implementations, the sample may contain an ultra-high dose of ascorbic acid from a patient who is using high doses of vitamins to relieve or alleviate symptoms of certain diseases. Without an anti-interference agent such as ascorbic acid oxidase, the results may be inaccurate. Therefore, in some implementations, as described herein, the slide contains an anti-interference agent in the gel layer or any other suitable layer of the slide.

[0133] In some implementations, a detection paradigm is used to analyze the dye on the microscope slide. The detection paradigm can be performed using one or more reflectance measurements. In one implementation, a reflectance measurement is used to detect the absorbance of the dye described herein.

[0134] In one implementation, light of a specific wavelength is directed to a detection area 128, and the reflected light, or reflectance density, is measured at that specific wavelength. Reflectance density (DR) is determined by reflectance. Reflectance density is equal to the logarithm of the reciprocal of reflectance.

[0135] In some embodiments, light is reflected from a titanium dioxide layer in the microscope slide. In some embodiments, the specific wavelength of light may be 540 nm, 670 nm, or both. In some embodiments, wavelengths near these values ​​or a range including these values ​​may be used, depending on the signal strength and / or interfering substances that may absorb light in the same spectrum.

[0136] In some implementations, wavelengths in the Soret and Q band regions may be used. Wavelengths may include those in the range of about 540 nm, such as, but not limited to, about 535 nm, about 536 nm, about 537 nm, about 538 nm, about 539 nm, about 541 nm, about 542 nm, about 543 nm, about 544 nm, or about 545 nm. In other embodiments, wavelength ranges such as, but not limited to, the following may be used: 530nm to 540nm, 539nm to 541nm, 538nm to 542nm, 537nm to 543nm, 536nm to 544nm, 535nm to 545nm, 540nm to 545nm, 535nm to 540nm, 535nm to 575nm, 530nm to 575nm, 540nm to 575nm, 550nm to 575nm, or 560nm to 575nm.

[0137] Similarly, in some embodiments, wavelengths of approximately 670 nm may be used, such as, but not limited to, approximately 665 nm, approximately 666 nm, approximately 667 nm, approximately 668 nm, approximately 669 nm, approximately 671 nm, approximately 672 nm, approximately 673 nm, approximately 674 nm, or approximately 675 nm. In other embodiments, wavelength ranges such as, but not limited to, the following may be used: 660 nm to 680 nm, 669 nm to 671 nm, 668 nm to 672 nm, 667 nm to 673 nm, 666 nm to 674 nm, 665 nm to 675 nm, 670 nm to 675 nm, and 665 nm to 670 nm.

[0138] In one implementation, the reflectance density is read by an automated analyzer such as, but not limited to, a VITROS analyzer. The endpoint reflectance density or reflectance can be quantified by the dye produced by the oxidation reaction.

[0139] The measurement time can vary depending on the analytical protocol or instrument used. However, typically, the time from sample application through the enzyme cascade to reflectance density quantification is approximately 5 to 10 minutes, approximately 4 to 6 minutes, approximately 3 to 7 minutes, approximately 2 to 8 minutes, less than approximately 10 minutes, less than approximately 9 minutes, less than approximately 8 minutes, less than approximately 7 minutes, less than approximately 6 minutes, or less than approximately 5 minutes. In one embodiment, the typical measurement time on a VITROS analyzer at 37°C is approximately 5 minutes.

[0140] Although the assay can be run at 37°C on the VITROS analyzer, other temperatures may also be used. For example, in some embodiments, the assay can be run at room temperature or at temperatures above or below 37°C.

[0141] In some implementations, components of the enzymatic cascade, such as proteases, fructose oxidases, peroxidases, dyes, and / or FADs, can be removed from the gel layer because they are not required for reading hemoglobin signals at 540 nm.

[0142] In some embodiments, a single microscope slide can be used to measure only %A1c. In other embodiments, separate microscope slides can be used to measure Hb and HbA1c separately to obtain derived %A1c results. Alternatively, all components required for determining Hb and HbA1c concentrations can be combined in a single microscope slide to obtain derived %A1c results.

[0143] The devices, systems, and methods described herein can use whole blood patient samples without dilution or pretreatment. In some embodiments, the whole blood is undiluted. Using whole blood saves time and resources compared to testing systems that require processed blood.

[0144] In some implementations, the devices, systems, and methods described herein can use a small amount of blood to measure HbA1c values. In clinical and diagnostic settings, blood sample size can be critical, especially when conducting large-scale testing.

[0145] Small amounts of blood can be between about 1 µL and about 10 µL, about 2 µL and about 8 µL, about 4 µL and about 6 µL, about 4 µL and about 5 µL, about 4 µL and about 10 µL, about 2 µL and about 5 µL, less than about 10 µL, less than about 8 µL, less than about 6 µL, or less than about 5 µL.

[0146] In some implementations, the devices, systems, and methods described herein can measure HbA1c values ​​in a shorter time frame compared to conventional methods. This may be referred to as rapid measurement time. In clinical and diagnostic settings, measurement time can be critical when considering time costs and instrument throughput.

[0147] The rapid measurement time can be approximately 1 minute to approximately 10 minutes, approximately 2 minutes to approximately 8 minutes, approximately 3 minutes to approximately 7 minutes, approximately 4 minutes to approximately 7 minutes, approximately 4 minutes to approximately 8 minutes, approximately 5 minutes to approximately 7 minutes, approximately 6 minutes to approximately 8 minutes, approximately 5 minutes to approximately 8 minutes, less than approximately 10 minutes, less than approximately 8 minutes, less than approximately 7 minutes, or less than approximately 6 minutes.

[0148] With rapid assay times, high-throughput systems employing the assays described herein can run more assays per time period, thereby generating greater gains per time period than conventional assays. In some embodiments, the high-throughput system can run approximately 300 assays / hour to approximately 400 assays / hour, approximately 350 assays / hour to approximately 400 assays / hour, approximately 350 assays / hour to approximately 450 assays / hour, approximately 300 assays / hour to approximately 500 assays / hour, approximately 300 assays / hour to approximately 600 assays / hour, at least approximately 300 assays / hour, at least approximately 350 assays / hour, at least approximately 375 assays / hour, or at least approximately 400 assays / hour.

[0149] The devices, systems, and methods described herein may have assay specificity, which is ensured by the production of a substrate (Fru-α-ValHis) through proteolytic hydrolysis of the N-terminal β-chain of hemoglobin and by desaccharification by a specific fructosyl peptide oxidase.

[0150] In some implementations, the devices, systems, and methods are unaffected by hemoglobin structural variants (HbS, HbC). Some commercially available assays are affected by HbS and HbC because they are antibody-based methods.

[0151] The method of using the microscopy slides described herein is also described.

[0152] In one embodiment, a single-slide method for detecting hemoglobin and glycated hemoglobin is described. The method may include contacting a spread layer of a microscope slide, as described herein, with a blood sample containing red blood cells. A lysing agent releases glycated hemoglobin from the red blood cells, a denaturing agent contacts the glycated hemoglobin to denature it, and a protease releases fructosyl peptides from the denatured glycated hemoglobin. The fructosyl peptides then reach the gel layer and contact fructosyl oxidase to release peroxides. The peroxidase and peroxides contact a detection agent to release and / or generate a detectable signal.

[0153] In some embodiments, the anti-interference agent reacts with any ascorbic acid in the blood sample to prevent sample bias. In some embodiments, the anti-interference agent is ascorbic acid oxidase.

[0154] The amount of hemoglobin is measured from a blood sample. This measurement includes reading the reflectance density of the sample using light of a first wavelength. Furthermore, the amount of glycated hemoglobin is measured from the blood sample. The glycated hemoglobin measurement includes detecting the reflectance density of a detectable signal from the sample under light of a second wavelength. In some embodiments, the second wavelength is different from the first wavelength. In one embodiment, the first wavelength is 540 nm and the second wavelength is 670 nm.

[0155] In some implementations, the first wavelength of light can be measured relatively early in the analysis, and the second wavelength of light can be measured later in the analysis after a delay time. The delay time can be approximately 30 seconds, approximately 40 seconds, approximately 50 seconds, approximately 60 seconds, approximately 2 minutes, approximately 3 minutes, approximately 4 minutes, approximately 30 seconds to approximately 1 minute, approximately 40 seconds to approximately 1 minute, approximately 30 seconds to approximately 2 minutes, approximately 30 seconds to approximately 3 minutes, or approximately 30 seconds to approximately 4 minutes. This delay time allows sufficient time for the reaction cascade to occur.

[0156] In another embodiment, a single-slide method for the direct detection of glycated hemoglobin is described. The method may include contacting a first membrane layer of a microscope slide, as described herein, with a blood sample containing red blood cells. A lysing agent releases glycated hemoglobin from the red blood cells, a denaturing agent contacts the glycated hemoglobin to denature it, and a protease releases fructosyl peptides from the denatured glycated hemoglobin. The fructosyl peptides then cross a second membrane layer, where they reach a third membrane layer and contact a fructosyl oxidase to release peroxides. The peroxidase and peroxides react with a detection agent to generate a detectable signal.

[0157] In some embodiments, the anti-interference agent reacts with any ascorbic acid in the blood sample to prevent sample bias. In some embodiments, the anti-interference agent is ascorbic acid oxidase.

[0158] The amount of glycated hemoglobin is then measured from the blood sample. In such embodiments, non-glycated hemoglobin is not measured. The glycated hemoglobin measurement involves detecting the reflectance density of a detectable signal in the sample. The glycated hemoglobin measurement involves detecting the reflectance density of a detectable signal from the sample under light of a specific wavelength. In some embodiments, this wavelength is 670 nm.

[0159] In some implementations, the microscopy slides described herein can have a lower unit manufacturing cost compared to conventional HbA1c measurement methods. The microscopy slides can reduce manufacturing costs by approximately 5% to approximately 10%, approximately 5% to approximately 20%, or approximately 10% to approximately 20%.

[0160] The microslides described herein can be produced by coating a continuous thin film layer onto a transparent support. Thus, microslides can be produced by applying a gel layer coating onto the support (114), then applying a masking layer onto the gel layer, applying an adhesive layer onto the masking layer, and applying a spreading layer onto the adhesive layer.

[0161] In other embodiments, once a coating has been formed through continuous film deposition, the coating is slit to a suitable width. The slit coating is then assembled into finished microscope slides by cutting the slits into individual slide-sized pieces, which can be mounted together with spacer webs into upper and lower slide mounts. This process can be performed on a slide assembly machine (SAM).

[0162] Individual slides can be packaged into a trolley for use on the main unit analyzer. The trolley can contain any number of slides suitable for the analyzer. In some embodiments, the trolley may have 50, 100, 200, at least 10, at least 15, at least 20, at least 50, or at least 100 slides. In other embodiments, the trolley may have 18, 50, or 60 slides.

[0163] In some implementations, inkjet deposition processes can be used to add spreader components. Spreader components added via inkjet deposition processes may include proteases, sodium nitrite, and / or calcium chloride.

[0164] In some embodiments, the microscope slide may include a spacer 130 between the support layer 114 and the lower slide mount 112. The spacer prevents damage to the microscope slide during assembly, particularly during the welding of the upper and lower slide mounts.

[0165] In some embodiments, the microscope slide has a thickness. The thickness of the microscope slide, which includes a spreading layer, an adhesive layer, a masking layer, a gel layer, optional spacer webs, and a support layer, is about 100 µm, about 200 µm, about 300 µm, about 400 µm, about 500 µm, or about 600 µm.

[0166] Example 1

[0167] Use a TiO2 masking layer to reduce optical interference from hemoglobin.

[0168] Six microscope slides are provided. Four of the slides contain TiO2 in the masking layer, and two do not. A blood sample is dropped onto the spread layer on each slide.

[0169] Figure 2The effect of the TiO2 masking layer is illustrated in the images. Slides 1 and 3 show the spot side of the TiO2 masking slide. Hemoglobin is clearly visible on the spot side of the slide. Slides 2 and 4 show the reading side of the TiO2 masked slide. Hemoglobin is excluded from the gel layer (reading side) of the slide.

[0170] In contrast, slides 5 and 6 show the spot side (slide 5) and read side (slide 6) of a microscope slide without TiO2 in the masking layer, respectively. Hemoglobin is easily visible on slide 6 (read side).

[0171] Therefore, including TiO2 in the masking layer will prevent any significant amount of hemoglobin from penetrating into the gel layer.

[0172] Figure 3A and 3B Figures 4A and 4B show the kinetics of a 10-order series of hemolytic products for the “control coating” (non-TiO2 masking layer) and the TiO2 masking layer, in the range of hemoglobin concentration from about 6 g / dL to about 20 g / dL.

[0173] The kinetics were recorded at 540 nm and 670 nm over a 5-minute time interval. Note the different scale bars in the figures. The control coating shows typical kinetic characteristics for the hemoglobin signal at 540 nm. The 540 nm response of the TiO2 masking layer slide indicates that the 540 nm signal has been masked because hemoglobin is excluded from the gel layer of the coating.

[0174] Similarly, the control coating exhibited typical kinetic characteristics in its hemoglobin signal at 670 nm. This data indicates the presence of a distinct hemoglobin signal at 670 nm, depending on the concentration of the hemoglobin being evaluated. This signal can cause optical interference in HbA1c measurements at 670 nm dye readings, necessitating a correction algorithm. The TiO2 in the masking layer significantly reduced the hemoglobin signal at 670 nm, thus preventing optical interference in HbA1c colorimetric measurements (read at 670 nm).

[0175] Figure 5 and Figure 6 The dose-response diagram of pure Fru-α-ValHis dipeptide in fluids with increased hemoglobin concentration is illustrated, comparing slides with and without TiO2 masking layers. Figure 5 In this study, the fluid ran on a TiO2-free glass slide. Data showed that with increasing hemoglobin concentration, the background signal (0.0 mM Fru-α-ValHis) at 670 nm increased, leading to a loss of the Δ signal range at the tested Fru-α-ValHis level. Figure 6In this study, fluid flowed on a glass slide containing a TiO2 masking layer. Data showed that the background signal (0.0 mM Fru-α-ValHis) at 670 nm remained unchanged with increasing hemoglobin concentration. Optical interference from hemoglobin at 670 nm was reduced and / or eliminated.

[0176] Example 2

[0177] HbA1c microslide test

[0178] Evaluation model %A1c fluid and whole blood patient sample HbA1c microscopy slide data. Figures 7A-D illustrate the reflectance density (DR) dynamics data at 670 nm to compare the signal generation of the model %A1c fluid with that of the %A1c patient sample.

[0179] The %A1c model fluid and %A1c patient samples had comparable %A1c. Except for sodium nitrite and protease, which were applied via inkjet deposition, all HbA1c microslide components were X-hopper coated.

[0180] Furthermore, coating with a higher concentration (coverage) of the denatured surfactant (N-lauroyl sarcosine, NLS) increased the kinetic response of both the %A1c model fluid and the %A1c patient samples. As shown in Figures 7A-D, the %A1c patient samples and the %A1c model fluid exhibited similar kinetic properties. Additionally, Figures 7A-D demonstrate good discriminatory power among the evaluated %A1c levels.

[0181] Figure 8A and 8B DR kinetics data at 670 nm are illustrated to compare model-generated fluid signals with %A1c patient sample signals. This data was generated using an x-hopper coating incorporating all components of the enzymatic cascade described herein.

[0182] The %A1c model fluid and the %A1c patient sample have comparable %A1c values. For example... Figure 8A and 8B As can be seen, the %A1c patient samples and the %A1c model fluid exhibit similar dynamic properties. Furthermore, Figure 8A and 8B The results show that there is good differentiation among the evaluated %A1c levels.

[0183] Figure 9 This illustrates the DR dose-response data at 670 nm for %A1c model fluid and whole blood %A1c patient samples. This data was obtained using... Figure 8A and 8BThe data shown were generated using the same coating. This data clearly demonstrates that the response curve given by the whole blood patient sample is identical to that of the %A1c model fluid made from purified glycated hemoglobin. This indicates that the microscope slide described herein can lyse red blood cells, denature and digest glycated hemoglobin to produce a substrate, thereby generating a colorimetric signal.

[0184] Figure 10 A graph illustrating the correlation between predicted %A1c results for patient samples obtained from HbA1c microscopy slides and specified HPLC %A1c values ​​is presented. A linear calibration model based on the HbA1c microscopy slide reflectance density at 670 nm and MicroTip %A1c reference values ​​was used to predict each of the six parallel microscopy measurements for each patient sample. The average of the predicted %A1c values ​​from the microscopy slides was compared with the BioRad Variant HPLC %A1c results. Figure 10 The results confirmed that the determination of %A1c by microscopic slides and the determination of %A1c by HPLC have a very strong correlation.

[0185] Based on the data obtained and studied in this embodiment, it is feasible to perform direct %A1c measurement using the microscope slides described herein.

[0186] Example 3

[0187] %A1c derived from HbA1c and hemoglobin component determinations

[0188] Here, HbA1c microscopy slides as described in this article are used in combination with hemoglobin microscopy slides to generate deduced %A1c results for patient samples.

[0189] Here, HbA1c microscopy measures the component of glycated hemoglobin, while hemoglobin microscopy alone measures the total hemoglobin component. These two results are used to calculate the derived %A1c test result.

[0190] Figure 11 This diagram illustrates the dose-response plot of hemoglobin components from the %A1c patient sample used in the previous section. The reflectance density at 540 nm is plotted against the Vitros MicroTip hemoglobin concentration results (in g / dL).

[0191] Figure 12 This diagram illustrates the correlation between predicted microslide hemoglobin component concentrations for patient samples and specified MicroTip reference hemoglobin values. A linear calibration model based on hemoglobin microslide reflectance at 540 nm and MicroTip hemoglobin reference values ​​was used to predict each of the six parallel hemoglobin microslide measurements for each patient sample. Figure 11The average predicted values ​​of hemoglobin components measured by microslide were compared with the reference values ​​of MicroTip hemoglobin. The microslide hemoglobin component determination and the MicroTip hemoglobin determination showed a good correlation.

[0192] Figure 13 The dose-response plot of the HbA1c component in the %A1c patient sample used in Example 2 is illustrated. The reflectance density at 670 nm is plotted against the MicroTip HbA1c component concentration (in g / dL).

[0193] Figure 14 This diagram illustrates the correlation between predicted HbA1c component concentrations on microslides for patient samples and specified MicroTip reference HbA1c component values. A linear calibration model based on HbA1c microslide reflectance at 670 nm and MicroTip HbA1c component reference values ​​was used to predict each of the six parallel HbA1c component measurements on microslides for each patient sample. Figure 13 The average predicted values ​​of HbA1c components from microslides were compared with the reference values ​​for HbA1c components from MicroTip. The determinations of HbA1c components from microslides and MicroTip showed a good correlation.

[0194] Figure 15 A graph illustrating the correlation between predicted %A1c values ​​from patient samples and BioRad Variant HPLC reference %A1c values ​​is presented. The %A1c values ​​from the microslides were obtained by generating the derived %A1c values ​​using the National Glycohemoglobin Standardization Project's "Master Equation" (see the following equation taken from VITROS HbA1c MicroTip Assay Instructions for Use, Publication No. J55871_EN (Version 2.0)) from hemoglobin fraction results (g / dL) and HbA1c fraction results (g / dL).

[0195]

[0196] The average %A1c value of the microscopic slides was compared with the HPLC %A1c reference value. The %A1c determination derived from the microscopic slides showed a good correlation with the HPLC %A1c determination.

[0197] Data from Example 3 confirms that it is feasible to derive %A1c measurements by combining the hemoglobin microscopy slide assay and the HbA1c microscopy slide assay as described herein.

[0198] Example 4

[0199] %A1c derived from HbA1c and hemoglobin component determinations

[0200] Here, a single microslide test element is used to generate hemoglobin spectral results and HbA1c enzymatic results to produce a derived %A1c result for the patient sample. This is referred to as a “dual assay” microslide test element. The microslide used does not contain titanium dioxide in the masking layer, as it would impede the ability to read the hemoglobin signal at 540 nm. The protease and sodium nitrite can be incorporated into the microslide via inkjet deposition or an X-hopper coating process.

[0201] Figure 16 and Figure 17 The dose-response data for the HbA1c component (670 nm) and hemoglobin component (540 nm) of the dual-measurement microscopy slide element are illustrated. The reflectance density (DR) dose-response data show that the dual-measurement microscopy slide element can record a dose-dependent BBI HbA1c fluid series at 670 nm, indicating that the enzymatic cascade is functional. The microscopy slide element also measures the hemoglobin spectral reading at 540 nm in a dose-dependent manner. As described in Example 3, the derived A1c% measurement is feasible using the "dual-measurement" microscopy slide element.

[0202] Example 5

[0203] Direct %A1c measurement

[0204] A whole blood sample was applied to a microscope slide as described herein. The enzymatic cascade described herein generates an oxidative dye that absorbs light at 670 nm, while the microscope slide uses a masking layer to filter out non-glycated hemoglobin. At 670 nm, light is reflected off titanium dioxide in the masking layer, and the reflectance density is read. Using a %A1c calibration curve, the reflectance density is directly calculated as %A1c.

[0205] Example 6

[0206] Derivation using a single sample %A1c

[0207] A whole blood sample is applied to a microscope slide as described herein, which does not contain titanium dioxide in its masking layer. The enzymatic cascade described herein generates an oxidative dye that absorbs light at 670 nm and allows for the measurement of non-glycated hemoglobin at 540 nm. The values ​​obtained from the measurements at 540 nm and 670 nm allow for the calculation of the derived %A1c.

[0208] Example 7

[0209] Derivation of %A1c using two samples on a single microscope slide

[0210] Two whole blood samples were applied to two separate regions of a microscope slide. One region contained titanium dioxide in its masking layer and, as described herein, an enzymatic cascade produced an oxidative dye that absorbed light at 670 nm. The other sample region did not contain titanium dioxide, allowing direct measurement of unglycated hemoglobin at 540 nm. The values ​​obtained from the measurements at 540 nm and 670 nm allowed for the calculation of the derived %A1c.

[0211] Example 8

[0212] Derivation of %A1c using two measurements on a single glass slide

[0213] A whole blood sample was applied to a microscope slide as described herein. The enzymatic cascade described herein generates an oxidative dye that absorbs light at 670 nm, while the microscope slide uses its masking layer to eliminate non-glycated hemoglobin signals. At 670 nm, light is reflected from below the masking layer using titanium dioxide, and the reflectance density is read. Additionally, at 540 nm, light is reflected from above the masking layer onto VtE beads in the titanium dioxide and spreading layers, and the reflectance density of hemoglobin is read. The values ​​obtained from measurements at 540 nm and 670 nm allow for the calculation of the derived %A1c.

[0214] Example 9

[0215] Reduce ascorbic acid interference

[0216] Two samples from a patient who had been taking large doses of ascorbic acid for anti-cancer effects were run. The first sample was run on a slide containing ascorbic acid oxidase in the gel layer, while the second sample was run on a slide without ascorbic acid oxidase.

[0217] The results from the first sample showed a higher %A1c value than those from the second sample.

[0218] Unless otherwise indicated, all figures used in the specification and claims to represent the amount and properties of components, such as molecular weight, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximate values, which may vary depending on the desired properties sought to be obtained by the invention. At least, and not attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques. Although the numerical ranges and parameters that set forth the broad scope of the invention are approximate values, the values ​​set forth in specific embodiments are reported as precisely as possible. However, any numerical value inherently contains a certain degree of error, which is necessarily caused by the standard deviation present in their respective test measurements.

[0219] Unless otherwise indicated herein or obviously contradicted by the context, the terms “a,” “an,” and “the,” and similar designations used in the context of describing the invention (particularly in the context of the appended claims) should be interpreted to cover both the singular and plural. The description of ranges of values ​​herein is intended only as a shorthand method of individually referring to each individual value falling within that range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were described separately herein. Unless otherwise indicated herein or obviously contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all instances or exemplary language (e.g., “such”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

[0220] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of the group or other elements present herein. For convenience and / or patentability reasons, it is contemplated that one or more members of a group may be included in a group or removed from a group. When any such inclusion or removal occurs, the specification shall be deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0221] This document describes certain embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will employ such variations as appropriate, and the inventors intend that the invention may be practiced in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equivalents to the subject matter recited in the appended claims as permitted by applicable law. Furthermore, unless otherwise indicated herein or clearly contradicted by the context, any combination of the foregoing elements with all possible variations is covered within the scope of this invention.

[0222] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be made are within the scope of the invention. Therefore, alternative configurations of the invention may be used in accordance with the teachings herein, by way of example and not limitation. Thus, the invention is not limited to what is strictly shown and described.

Claims

1. A glass slide, the glass slide comprising: A stack of membrane layers, the stack comprising, from bottom to top: A first membrane layer comprising a cross-linked gel, wherein the cross-linked gel comprises a detection agent, a fructose oxidase, an anti-interference agent, and a peroxidase; A second film layer comprising a hardened gel and a reflective material portion comprising titanium dioxide, the reflective material portion being configured to generate a signal including a first wavelength of 540 nm for measuring Hb above the reflective material portion and a second signal including a second wavelength of 670 nm for measuring fructosyl oxidase below the reflective material portion, and The third membrane layer comprises polymer beads, a pyrolysis agent, a denaturant, and a protease. The hardened gel and the titanium dioxide are combined in such a way that they form a sieve that allows the Fru-α-ValHis peptide to pass through the second membrane layer while preventing larger molecular weight proteins from passing through the second membrane layer.

2. The glass slide according to claim 1, wherein the pyrolysis agent is a detergent.

3. The slide according to claim 2, wherein the detergent is selected from octylphenol ethoxylate (TRITON X-100), TWEEN (TWEEN 20), sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ether (POE), and NONIDET P-40 (NP-40).

4. The glass slide according to claim 3, wherein the detergent is octylphenol ethoxylate.

5. The glass slide according to claim 1, wherein the denaturant is a surfactant.

6. The slide according to claim 5, wherein the denaturing agent is one or more of sodium nitrite or N-lauroyl sarcosine (NLS).

7. The slide according to claim 1, wherein the protease is a metalloproteinase.

8. The glass slide according to claim 7, wherein the third film layer further comprises calcium.

9. The slide according to claim 8, wherein the protease is a neutral protease.

10. The slide according to claim 1, wherein the protease is an endopeptidase or an exopeptidase.

11. The slide according to claim 10, wherein the protease is selected from proteinase K, streptinase E, protease XVII, protease XXI, aminopeptidase, carboxypeptidase, thermophilic protease and subtilisin.

12. The slide according to claim 1, wherein the peroxidase is horseradish peroxidase.

13. The slide according to claim 1, wherein the detection reagent is selected from sodium N-carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine (DA-64), N,N,N'N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triamino-triphenylmethane hexasodium salt (TPM-PS), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenothiazine sodium (DA-67) and 2-(3,5-dimethoxy-4-hydroxyphenol)-4,5-bis-(4-dimethylaminophenyl)imidazole.

14. The glass slide according to claim 1, wherein the third film layer further comprises a layer having particles with a diameter of about 25 µm.

15. The slide according to claim 1, wherein the third film layer further comprises an oxidase cofactor and a surfactant.

16. The slide according to claim 15, wherein the oxidase cofactor is flavin adenine dinucleotide (FAD).

17. The slide according to claim 15, wherein the fructosyl oxidase is specific for Fru-α-ValHis peptide or Fru-α-Val amino acid.

18. The glass slide according to claim 1, wherein the first film layer is in direct contact with the lower surface of the second film layer.

19. The glass slide according to claim 18, wherein the second film layer is in direct contact with the lower surface of the third film layer.

20. The glass slide according to claim 1, wherein the anti-interference agent is ascorbic acid oxidase.

21. A single-slide method for detecting hemoglobin and glycated hemoglobin, the method comprising: a) Provide glass slides; The glass slide comprises: A stack of membrane layers, the stack comprising, from bottom to top: A first membrane layer comprising a cross-linked gel, wherein the cross-linked gel comprises a detection agent, a fructose oxidase, an anti-interference agent, and a peroxidase; A second film layer comprising a hardened gel and a reflective material portion comprising titanium dioxide, the reflective material portion being configured to generate a signal comprising a first wavelength of 540 nm for measuring Hb above the reflective material portion and a second signal comprising a second wavelength of 670 nm for measuring fructosyl oxidase below the reflective material portion. The hardening gel and the titanium dioxide are combined in such a way that they form a sieve that allows Fru-α-ValHis peptides to pass through the second membrane layer while preventing larger molecular weight proteins from passing through the second membrane layer. The third membrane layer comprises polymer beads, a pyrolysis agent, a denaturant, and a protease. b) Contact the third membrane layer of the slide with an unlysaturated blood sample containing red blood cells, wherein the lysing agent releases glycated hemoglobin from the red blood cells, wherein the denaturing agent contacts the glycated hemoglobin to denature the glycated hemoglobin, and wherein the protease releases fructosyl peptides from the denatured glycated hemoglobin, wherein the fructosyl peptides reach the first membrane layer and contact the fructosyl oxidase to release peroxides, and wherein the peroxidase and the peroxides contact the detection agent to release a detectable signal; c) Measuring the amount of hemoglobin from the blood sample, wherein the measurement of the amount of hemoglobin includes reading the sample reflectance density from the slide under light of a first wavelength; and d) Measuring the amount of glycated hemoglobin from the blood sample, wherein the measurement of the amount of glycated hemoglobin includes detecting the reflectance density of a detectable signal from the sample under light of a second wavelength, wherein the second wavelength of light is different from the first wavelength of light.

22. The method according to claim 21, wherein the pyrolysis agent is a detergent selected from octylphenol ethoxylate (TRITON X-100), TWEEN (TWEEN 20), sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ether (POE), and NONIDET P-40 (NP-40).

23. The method of claim 22, wherein the detergent is octylphenol ethoxylate.

24. The method of claim 21, wherein the denaturing agent is one or more of sodium nitrite or N-lauroyl sarcosine (NLS).

25. The method of claim 21, wherein the protease is a metalloproteinase, an endopeptide, or an exopeptide.

26. The method of claim 25, wherein the metalloproteinase is a neutral protease.

27. The method of claim 21, further comprising contacting the fructose-based peptide with an oxidase cofactor, wherein the oxidase cofactor is flavin adenine dinucleotide (FAD).

28. The method according to claim 21, wherein the peroxidase is horseradish peroxidase.

29. The method of claim 21, wherein the detection agent is a leuco dye.

30. A single-slide method for directly detecting glycated hemoglobin, the method comprising: a) Provide the glass slide according to claim 1; b) Contacting the third membrane layer of the slide with a blood sample containing red blood cells, wherein the lysing agent releases glycated hemoglobin from the red blood cells, wherein the denaturing agent contacts the glycated hemoglobin to denature the glycated hemoglobin, and wherein the protease releases fructosyl peptides from the denatured glycated hemoglobin, wherein the fructosyl peptides cross the second membrane layer, wherein the fructosyl peptides reach the first membrane layer and contact the fructosyl oxidase to release peroxide, and wherein the peroxidase and the peroxide contact the detection agent to release a detectable signal; and c) Measuring the amount of glycated hemoglobin from the blood sample, wherein the measurement of the amount of glycated hemoglobin includes detecting the reflectance density of a detectable signal in the sample.

31. The method of claim 30, wherein the fructose-based peptide passes through the cross-linked gel.

32. The method of claim 30, wherein the protease is a metalloproteinase, an endopeptide, or an exopeptide.

33. The method of claim 32, wherein the metalloproteinase is a neutral protease.

34. The method of claim 30, further comprising contacting the fructose-based peptide with an oxidase cofactor, wherein the oxidase cofactor is flavin adenine dinucleotide (FAD).

35. The method of claim 30, wherein the peroxidase is horseradish peroxidase.

36. The method of claim 30, wherein the detection agent is a leuco dye.

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