Glycated hemoglobin measurement

Abstract

This specification describes devices, systems, and methods used to measure glycated hemoglobin.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) 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 disclosures of which are incorporated herein by reference.

[0002] (Technical field) Described herein are devices, systems and methods used to measure glycated hemoglobin. Summary of the Invention [Means for solving the problem]

[0003] Described herein generally are devices, systems, and methods used for hemoglobin measurement, particularly for measuring glycated hemoglobin. Determining glycated hemoglobin levels in patient samples is an important component in diagnosing type 1 diabetes, type 2 diabetes, and gestational diabetes, as it allows for estimation of an individual's average blood glucose level over a period of time (e.g., three months).

[0004] In some embodiments, this glycated hemoglobin measurement can be of whole blood, which can be human or animal whole blood, but measurements can also be made of various components of blood, so long as the hemoglobin component is present.

[0005] Generally, measurements can be performed using microslides, such as microslide test elements, or simply dry slide test elements. These microslides can be used in automated analyzers. The microslides can be single slides, allowing all analyses to be completed with one or a few drops of sample and a single slide, rather than multiple slides. In some embodiments, multiple measurements can be performed on a single slide.

[0006] The microslides described herein can include a stack of film layers including, from bottom to top, a first film layer comprising a cross-linked gel, where the cross-linked gel comprises a detection agent, a fructosyl oxidase, and a peroxidase; a second film layer comprising the first gel; and a third film layer comprising a lysing agent, a denaturant, and a protease. In some embodiments, the first film layer is a gel layer, the second film layer is a mask layer, and the third film layer is a spreading layer. In some embodiments, the microslide can include an adhesive layer or sub-layer between the mask layer and the spreading layer.

[0007] The microslides described herein can include a stack of film layers including, from bottom to top, a first film layer comprising a crosslinked gel, the crosslinked gel comprising a detection agent, a fructosyl oxidase, an anti-interference agent, and a peroxidase; a second film layer comprising the first gel; and a third film layer comprising a lysis agent, a denaturant, 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 microslide can include an adhesive layer or sub-layer between the masking layer and the spreading layer.

[0008] The microslides described herein can include a stack of film layers including, from bottom to top, a gel layer, a masking layer, and a spreading layer. In other embodiments, the microslides described herein can include a stack of film layers including, from bottom to top, a gel layer, a masking layer, an adhesive layer or sub-layer, and a spreading layer. In some embodiments, the first layer, or gel layer, includes a gel. In some embodiments, the second film layer, the masking layer, includes a second gel. In some embodiments, the third film layer, the spreading layer, includes a lysing agent, a denaturant, and a protease. In some embodiments, the adhesive layer or sub-layer is included as the third layer, and the spreading layer is the fourth layer.

[0009] In some embodiments, the gel can be a cross-linked gel. Other layers may be included in the microslide. In some embodiments, the cross-linked gel includes a detection agent, fructosyl oxidase, and peroxidase.

[0010] The first film layer can further comprise an oxidase cofactor and a surfactant. In some embodiments, the oxidase cofactor is flavin adenine dinucleotide (FAD). The fructosyl oxidase can be specific for glycated amino acids such as Fru-α-ValHis peptide or Fru-α-Val. In some embodiments, the anti-interference agent is ascorbic acid oxidase (AAO).

[0011] In some embodiments, the disclosing agent is a leuco dye, such as a blue leuco dye. Ru-It may be selected from the group consisting of 4,4'-bis(dimethylamino)-diphenylamine sodium (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.

[0012] In some embodiments, the peroxidase is horseradish peroxidase.

[0013] In some embodiments, the second film layer further comprises a reflective material portion. The reflective material portion can include a metal salt. In one embodiment, the metal is titanium, such as, but not limited to, titanium dioxide (TiO2).

[0014] In some embodiments, the third film layer further comprises calcium.

[0015] In some embodiments, the third film layer comprises a porous layer containing latex particles. In some embodiments, the latex particles can be formed from vinyl toluene-co-methacrylic acid copolymer (VtE). The particles, or particles sometimes referred to as beads, can have a median particle size of less than about 25 μm. In one embodiment, the particles can have a median particle size of 25 μm. In other embodiments, the particles can 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.

[0016] In some embodiments, the dissolution agent is a detergent. The detergent can be selected from the group consisting of octylphenol ethoxylate (TRITON® X-100, Union Carbide Corporation, New York), TWEEN® (ICI Americas Inc, Delaware) (TWEEN 20), sodium dodecyl sulfate (SDS), cetyltrimethylammonium 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.

[0017] In some embodiments, the denaturant is an oxidizing agent or a surfactant. The denaturant can be one or more of sodium nitrite or N-lauroyl sarcosine (NLS).

[0018] In some embodiments, the protease is a metalloproteinase and / or a neutral protease. The protease can be an endoprotease or an exoprotease. In other embodiments, the protease is selected from the group consisting of proteinase K, pronase E, protease XVII, protease XXI, aminopeptidase, carboxypeptidase, thermolysin, batyrolysin, microbial metalloproteinase, peptidase K, endoproteinase K, chymotrypsin, chymotrypsin C, glutamyl endopeptidase, peptidyl lys metalloendopeptidase, protease from Bacillus genus, leucyl aminopeptidase, and subtilisin.

[0019] In some embodiments, the third film layer is in direct contact with the top surface of the second film layer. In other embodiments, the second film layer is in direct contact with the top surface of the first film layer.

[0020] In some embodiments, the spreading layer is in direct contact with the top surface of the masking layer, while in other embodiments, the spreading layer is in direct contact with the top surface of the adhesive layer.

[0021] Also described herein are single-slide methods for detecting hemoglobin and glycated hemoglobin, including: a) providing a slide as described herein; b) contacting a third film layer of the slide with an unlysed blood sample containing red blood cells, wherein a lysing agent releases glycated hemoglobin from the red blood cells; a denaturing agent contacts and denatures the glycated hemoglobin; a protease releases fructosyl peptides from the denatured glycated hemoglobin; the fructosyl peptides reach the first film layer and contact fructosyl oxidase and FAD cofactor to generate peroxides; and the peroxidase and peroxides are denatured. contacting the detecting agent with the detecting agent to emit a detectable signal, c) measuring the amount of hemoglobin from the blood sample, wherein measuring the amount of hemoglobin comprises reading a reflectance density of the sample from the slide at a first wavelength of light, and d) measuring the amount of glycated hemoglobin from the blood sample, wherein measuring the amount of glycated hemoglobin comprises detecting a reflectance density from the detecting agent at a second wavelength of light, the second wavelength of light being different from the first wavelength of light. In some embodiments, the detecting agent is an oxidation dye.

[0022] In some embodiments, the unprocessed blood sample can be an unlysed blood sample. In some embodiments, the unprocessed blood sample can be blood that has been subjected to an anticoagulant rather than a lysing agent. In some embodiments, the anticoagulant is an anticoagulant. In some embodiments, the unprocessed blood can be whole blood.

[0023] In some embodiments, the first wavelength of light is 540 nm and the second wavelength of light is 670 nm.

[0024] In some embodiments, the method further comprises contacting the fructosyl peptide with an oxidase cofactor, such as flavin adenine dinucleotide (FAD).

[0025] Also described herein are single-slide methods for directly detecting glycated hemoglobin, including: a) providing a microslide having a second film layer including a reflective material portion; b) contacting a third film layer of the slide with a blood sample including untreated red blood cells, wherein a lysing agent releases glycated hemoglobin from the red blood cells, a denaturing agent contacts and denatures the glycated hemoglobin, a protease releases fructosyl peptides from the modified glycated hemoglobin, the fructosyl peptides traverse the second film layer, the fructosyl peptides reach the first film layer and contact fructosyl oxidase and FAD cofactor to generate peroxide, and the peroxidase and peroxide contact a detecting agent to emit a detectable signal; and c) measuring the amount of glycated hemoglobin from the blood sample, wherein measuring the amount of glycated hemoglobin includes detecting the reflectance concentration of an oxidation dye in the blood sample.

[0026] In some embodiments, the reflective material comprises a metal salt.

[0027] The whole blood sample is able to traverse the third film layer due to the porosity created by the latex particles in the third layer. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows the layers incorporated into the microslides described herein. [Figure 2] FIG. 2 shows a comparison of a slide in which the masking layer contains TiO2 and a slide in which the masking layer does not contain TiO2. [Figure 3A]FIG. 3A shows the signal at 540 nm for a masking layer without TiO2 ("control") and a masking layer with TiO2. [Figure 3B] FIG. 3B shows the signal at 540 nm for a masking layer without TiO2 ("control") and a masking layer with TiO2. [Figure 4A] FIG. 4A shows the signal at 670 nm for a masking layer without TiO2 ("control") and a masking layer with TiO2. [Figure 4B] FIG. 4B shows the signal at 670 nm for a masking layer without TiO2 ("control") and a masking layer with TiO2. [Figure 5] FIG. 5 shows the dose response of Fru-VH substrate in the presence of increasing hemoglobin (Hb) concentrations for slides without TiO 2 in the masking layer. [Figure 6] FIG. 6 shows the dose response of Fru-VH substrate in the presence of increasing hemoglobin (Hb) concentrations for slides containing TiO 2 in the masking layer. [Figure 7A] Figure 7A shows the kinetic data for the %Alc model fluid and %Alc patient sample at 670 nm over 5 minutes in the analyzer at 37° C. Protease and sodium nitrite were inkjet deposited and dried onto the completed microslide. [Figure 7B] Figure 7B shows the kinetic data for the %Alc model fluid and %Alc patient sample at 670 nm over 5 minutes in the analyzer at 37° C. Protease and sodium nitrite were inkjet deposited and dried onto the completed microslide. [Figure 7C] Figure 7C shows the kinetic data for the %Alc model fluid and %Alc patient sample at 670 nm over 5 minutes in the analyzer at 37° C. Protease and sodium nitrite were inkjet deposited and dried onto the completed microslide. [Figure 7D]Figure 7D shows the kinetic data for the %Alc model fluid and %Alc patient sample at 670 nm over 5 minutes in the analyzer at 37° C. Protease and sodium nitrite were inkjet deposited and dried onto the completed microslide. [Figure 8A] Figure 8A shows the kinetic data of the % Alc model liquid and % Alc patient sample at 670 nm over 5 minutes in the analyzer at 37° C. All components are incorporated by an x-hopper coating process. [Figure 8B] Figure 8B shows the kinetic data of the % Alc model liquid and % Alc patient sample at 670 nm over 5 minutes in the analyzer at 37° C. All components are incorporated by an x-hopper coating process. [Figure 9] Figure 9 shows the reflectance concentration dose response data of the % Alc model liquid and % Alc patient sample at 670 nm over a 5 minute period in the analyzer at 37° C. All components are incorporated by an x-hopper coating process. [Figure 10] FIG. 10 shows the correlation plot between Microslide %A1c and Biorad Varian HPLC %A1c. [Figure 11] Figure 11 shows the dose response of microslide hemoglobin component patient samples. [Figure 12] FIG. 12 shows a correlation plot between the microslide hemoglobin component assay and the microchip reference hemoglobin component assay. [Figure 13] FIG. 13 shows the dose response of Microslide HbA1c component patient samples. [Figure 14] FIG. 14 shows a correlation plot between the microslide HbA1c component assay and the microchip reference HbA1c component assay. [Figure 15] FIG. 15 shows the correlation plot between the microslide-derived %A1c assay and the HPLC reference %A1c assay. [Figure 16]FIG. 16 shows the HbA1c enzyme 670 nm dose response plot for the dual assay microslide test element. [Figure 17] FIG. 17 shows the hemoglobin spectral 540 nm dose-response plot for the dual assay microslide test element. DETAILED DESCRIPTION OF THE INVENTION

[0029] The use of a thin film test element, a microslide, that performs an enzyme cascade to measure glycated hemoglobin concentration using a patient sample by direct means (%A1c measurement only) or by derived calculation (HbA1c and hemoglobin measurement to obtain a %A1c result) is described. In some embodiments, the patient sample is a whole blood sample, such as an unprocessed whole blood sample. In some embodiments, the unprocessed blood sample can be an unlysed blood sample. In some embodiments, the unprocessed blood sample can be blood that has been subjected to an anticoagulant but not to a lysing agent. In some embodiments, the anticoagulant is an anticoagulant. In some embodiments, the unprocessed blood can be whole blood.

[0030] The blood sample can be human or animal blood. In some embodiments, the blood can be mammalian blood. Mammals can include, but are not limited to, humans, horses, camels, dogs, cats, cows, bears, rodents, sheep, goats, pigs, etc. Other animal blood can also be used, such as reptile, fish, and avian blood.

[0031] In some embodiments, various components or fractions of a patient's blood sample can also be measured, so long as the hemoglobin component is present.

[0032] Measurements are accomplished using the devices, systems, and methods described herein. Generally, measurements can be performed using a microslide, such as a dry microslide. The microslide can be a single slide, whereby an entire analysis can be completed using a single drop of sample and a single slide, rather than multiple slides.

[0033] The microslides described herein can be utilized in automated analytical systems or other types of mainframe analyzers. These types of instruments, in some embodiments, can process hundreds or thousands of sample analyses per workday. In one embodiment, the microslides can be used with current VITROS mainframe analyzers (5,1 FS, 4600 Chemistry System, 5600 Integrated System) and future VITROS analyzers as well. Additionally, the microslides described herein can be used in systems manufactured by Abbott Laboratories, Beckman Coulter, Baxter, Genprobe, Roche Diagnostics, and Siemens.

[0034] However, in other embodiments, the microslides can be utilized in non-automated or semi-automated systems. In some embodiments, the microslides can be used in a sample-by-sample scenario and / or can be manually loaded.

[0035] In some embodiments, the microslide test elements described herein can incorporate components of an enzyme cascade that can result in the production of a colorimetric signal that is directly related to the concentration of glycated hemoglobin (as %A1c) in a patient sample.

[0036] Hemoglobin glycation is commonly determined in the industry by measuring both hemoglobin (Hb) and glycated hemoglobin (HbA1c) concentrations and expressing the ratio (derived %A1c). This requires two sets of standards to create separate calibration curves for Hb and HbA1c measurements. Alternatively, assays can be calibrated using a liquid with a known %A1c as a standard to directly provide the %A1c of unknown patient samples.

[0037] The devices, systems, and methods described herein can use available microslides in either assay format. In one embodiment, all components necessary to determine Hb and HbA1c concentrations can be incorporated into a single test element, resulting in a derived %A1c result. In such an embodiment, Hb and HbA1c can each be determined at different detection wavelengths from a single slide, and the test can be performed from a single whole blood measurement event and can be performed using current microslide protocols.

[0038] In another embodiment, a second option is to measure the Hb and HbA1c concentrations separately using separate microslides to obtain a derived %A1c result. In such an embodiment, the Hb and HbA1c concentrations can each be determined at different detection wavelengths from individual test slides within a single test element, and the test can be performed from two whole blood measurement events and can be performed using current microslide protocols.

[0039] Additionally, in another embodiment, %A1c can be measured directly using a single microslide. In such an embodiment, %A1c can be determined from a single microslide at a single detection wavelength. Measurements can be made from a single whole blood measurement event and can be made using current microslide protocols.

[0040] In some embodiments, the devices, systems and methods can use an enzyme cascade to determine HbA1c as %A1c. The enzyme cascade used in the microslides described herein can be: [ka] is.

[0041] In some embodiments, the cascade can be used to measure glycated hemoglobin directly (as %A1c), however, the cascade can also be used to measure derived %A1c.

[0042] Generally, methods for determining direct or derived %A1c involve applying an unprocessed whole blood sample to a microslide described herein. In some embodiments, the microslide may include more than one sample location and may require more than one blood sample.

[0043] A lytic detergent can lyse red blood cells in a blood sample, thereby liberating glycated hemoglobin. A second detergent can denature the glycated hemoglobin, thereby providing access to proteolytic cleavage sites. Next, proteases cleave the N-terminal portion of the hemoglobin beta chain, liberating a glycated dipeptide (fructosyl-α-valyl-histidine-Fru-α-ValHis). The glycated dipeptide is deglycosylated by oxidation using fructosyl peptide oxidase (FPOX) and flavin adenine dinucleotide (FAD), which generates hydrogen peroxide (H2O2). H2O2 and horseradish peroxidase (HRP) oxidize a leuco dye, resulting in a colorimetric signal at 670 nm. The concentration of glycated hemoglobin is directly proportional to the reflectance concentration of the dye formed. In some embodiments, the hemoglobin signal is read at 540 nm and a determination of the hemoglobin component at 540 nm and a determination of glycated hemoglobin at 670 nm can be used to determine a derived %A1c value.

[0044] In some embodiments, the microslide can include at least a first film layer, a second film layer, and a third film layer. The microslide can include more layers. In some embodiments, the first film layer can include cross-linked gelatin or gel, and the cross-linked gel can include a detection agent, fructosyl oxidase, peroxidase, and optionally an anti-interference agent. In some embodiments, the second film layer can include gelatin, gel, or a cross-linked gel or cross-linked gelatin. In some embodiments, the third film layer can include a lysis agent, a denaturant, and / or a protease.

[0045] Microslide 100 can include a stack of film layers or simply a stack of layers. A stack of film layers can include a gel layer 102, a masking layer 104, an adhesive layer 106, and a spreading layer 108, as shown in FIG. 1 . In some embodiments, microslide 100 can include an upper slide mount 110, a lower slide mount 112, or both. In some embodiments, when formed, the layers can be constructed on a support layer 114. In some embodiments, adhesive layer 106, masking layer 104, and gel layer 102 can be combined as a reagent layer.

[0046] In some embodiments, the support layer 114 can be formed of polyethylene terephthalate or other suitable transparent polymeric material that allows for layers to be applied or coated thereon.

[0047] In some embodiments, upper slide mount 110 and lower slide mount 112 are formed from polystyrene or other suitable polymeric material.

[0048] The layers and mounts can be combined to form a microslide that has a square or approximately rectangular top surface 116 and bottom surface 118. In some embodiments, the top surface 116 and bottom surface 118 can have other shapes, such as, but not limited to, a triangle, a pentagon, a hexagon, a heptagon, an octagon, a circle, an oval, or other rectangular or circular shapes.

[0049] In some embodiments, a microslide can include at least one notch or keying surface. The notch or keying surface can be used to assist in stacking multiple microslides and / or loading one or more microslides into an analytical instrument. In one embodiment, microslide 100 can include notch 120. While notch 120 is shown as having a rectangular shape, in other embodiments, notch 120 can be virtually any shape that allows for stacking and / or loading.

[0050] Additionally, the microslide 100 may include a window 122 on the top surface 116 and / or the bottom surface 118. The window 122 may be surrounded by a frame 124. However, in some embodiments, a frame is not included and the window 122 may extend to the edge of the microslide.

[0051] The microslide 100 can include a sample area 126 within a window 122 on the upper surface 116. The sample area 126 can serve as a location for sample application. On the lower surface 118, a detection area 128 can be present within the window 122. The detection area 128 can serve as a location for detection using an analyzer.

[0052] The layers of the microslide that the sample moves through are now described. The spreading layer 108 can be the first layer that the sample encounters. The spreading layer 108 can include polymer beads, a binder, a buffer, at least one surfactant, a divalent cation salt, sodium nitrite, a protease, alcohol, and water.

[0053] In some embodiments, the divalent cation salt can be calcium chloride or any compound capable of binding EDTA.

[0054] In some embodiments, tert-butyl alcohol may be present when forming or applying the spread layer, but not after drying.

[0055] In some embodiments, the polymeric beads can include, but are not limited to, acrylic beads, such as vinyl toluene-co-methacrylic acid copolymer beads (VtE beads). The function of the beads can be to form pores in the spreading layer that allow red blood cells to enter the coating. The beads can also provide a white reflective surface, promote uniform sample spreading, and function to capture interferents such as heme by-products, catalase, and triglycerides.

[0056] The beads can have an average diameter large enough to allow red blood cells to penetrate the coating, in some embodiments, the diameter is greater than about 5 μm, greater than about 10 μm, greater than about 50 μm, greater than about 80 μm, between about 20 μm and about 100 μm, between about 20 μm and about 30 μm, between about 10 μm and about 40 μm, between about 10 μm and about 100 μm, between about 20 μm and about 25 μm, between about 50 μm and about 100 μm, between about 25 μm and about 30 μm, less than about 100 μm, less than about 80 μm, less than about 50 μm, less than about 40 μm, or less than about 30 μm.

[0057] The pores produced by the beads can have a pore size greater than about 5 μm, greater than about 10 μm, greater than about 20 μm, between about 20 μm and about 30 μm, between about 10 μm and about 40 μm, between about 20 μm and about 25 μm, between about 25 μm and about 30 μm, less than about 50 μm, less than about 40 μm, or less than about 30 μm. In one embodiment, the pore size is about 25 μm.

[0058] The binder, which can function to promote layer cohesion, can be a latex. In one embodiment, the latex can have a percent solids of about 30% of the molecular weight of monomer (MWM) latex in the final product. In some embodiments, the latex includes a biocide, such as, but not limited to, nipacide. In other embodiments, alternative binders, such as, but not limited to, polyacrylamide (I100), can be utilized.

[0059] The buffering agent functions 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 buffering agent can be an acid or a base, as appropriate. In one embodiment, the buffer is 3-(N-morpholino)propanesulfonic acid (MOPS). Other buffering agents can include, but are not limited to, sodium bicarbonate, calcium carbonate, potassium phosphate, tris(hydroxymethyl)aminomethane (TRIS), bicine, Bis-TRIS, TES, HEPPS (EPPS), and the like, or combinations thereof.

[0060] In some embodiments, the method can include a first surfactant and a second surfactant. The first surfactant can be a lysing agent. The lysing agent can lyse red blood cells and release hemoglobin and glycated hemoglobin. The lysing agent can be a detergent.

[0061] In some embodiments, the detergent can be selected from octylphenol ethoxylate (TRITON X-100), TWEEN (TWEEN 20), sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ethers (POEs), NONIDET P-40 (NP-40), or combinations thereof. In one embodiment, the detergent is an octylphenol ethoxylate, such as TRITON X-100.

[0062] In some embodiments, the second surfactant can be a denaturant that denatures hemoglobin. The denaturant can expose sites on hemoglobin for proteolysis. The denaturant can allow heme oxidation in a single oxidation state and aid in the conversion of hemoglobin forms (oxy, deoxy, carboxy) to a single spectral form.

[0063] In one embodiment, the denaturant can include N-lauroyl sarcosine (NLS). In some embodiments, the second surfactant can also include a denaturant aid or oxidizing agent, such as sodium nitrite. The denaturant aid can help promote denaturation by NLS by coordinating with the iron in heme.

[0064] In one embodiment, one or more denaturing agents present in the coating are capable of modifying glycated hemoglobin to make it accessible to the desired protease cleavage site.

[0065] In some embodiments, the spread layer 108 may optionally include additional surfactants to initiate the enzyme cascade.

[0066] Sodium nitrite can be present in molar excess. In some embodiments, sodium nitrite (NaNO) can be present at about 5-10 times the total hemoglobin concentration. In some embodiments, sodium nitrite can act to oxidize heme to the ferrous state (+3).

[0067] Furthermore, the combination of a denaturing surfactant, sodium nitrite, and hemoglobin can create a single spectral form of hemoglobin that can be read at 540 nm.

[0068] The protease can be a neutral protease. The protease can be a metalloprotease. The protease can be an endoprotease or an exoprotease. The protease can cleave from the N-terminus of the hemoglobin β subunit or chain to produce Fru-α-ValHis. Fru-α-ValHis can be a substrate for the fructosyl oxidase contained in the gel layer described herein.

[0069] In some embodiments, the protease can be proteinase K, pronase E, protease XVII, protease XXI, aminopeptidase, carboxypeptidase, thermolysin, subtilisin, or a combination thereof.

[0070] The Fru-α-ValHis dipeptide can be of sufficiently small molecular weight so that it can easily pass through the adhesive and masking layers and enter the gel layer described herein.

[0071] The calcium component may be essential for protease activity. In some embodiments, the calcium in the microslide can protect the protease from the EDTA anticoagulant in the blood collection tube. In some embodiments, the calcium source is calcium chloride (CaCl). In other embodiments, the calcium chloride is calcium chloride dihydrate.

[0072] The calcium chloride present in the spread layer can act to protect the activity of the protease. In some embodiments, clinical collection tubes for HbA1c measurements are EDTA plasma tubes. Without calcium chloride, EDTA can bind calcium and zinc from the protease, significantly reducing the proteolytic activity of the protease.

[0073] The spread layer solvent can be methanol, ethanol, tertiary butyl alcohol, etc., or combinations thereof. In one embodiment, the alcohol is about 97 w / w tertiary butyl alcohol.

[0074] The adhesive layer 106 directly below the spreading layer 108 may contain adhesive substances, surfactants, and / or solvents. The Fru-α-ValHis dipeptide produced in the spreading layer can easily pass through the adhesive layer 106.

[0075] In some embodiments, the adhesive can function to promote adhesion between the spreading layer 108 and the masking layer 104. In one embodiment, the adhesive is polyisopropylacrylamide (1100). In other embodiments, the adhesive can be polyvinylpyrrolidone (PVP). In some embodiments, the PVP can have a k90 chain length, a k30 chain length, a k15 chain length, or a combination thereof.

[0076] The adhesive layer surfactant can function 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 can be a combination of polyisopropylacrylamide and octylphenol ethoxylate.

[0077] The solvent for the adhesive layer 106 can be ethanol, isopropyl alcohol, methanol, t-butyl alcohol, acetone, or a combination thereof. In one embodiment, the adhesive layer solvent can be acetone. In some embodiments, if ethanol is used, a surfactant may not be necessary.

[0078] The masking layer 104 can include a gel, at least one buffer, a pigment material, a dispersant, a surfactant, a hardener, and / or a solvent / diluent. In some embodiments, the gel is gelatin and / or a hardened gel.

[0079] The gel can promote layer cohesion, promote capillary forces upon rewetting, and / or provide a size exclusion mechanism, which in some embodiments can exclude high molecular weight interferents (upon crosslinking / curing).

[0080] In some embodiments, the gel is a hardened gel. In one embodiment, the gel is Gel-RC Rousselot Dub Pig Dia Type 56 or 275 Bloom Type A NF Porcine Skin Gelatin.

[0081] In some embodiments, the pigment material can create reflective areas within the masking layer. The pigment material can provide a white, reflective surface and act to capture or mask interferents such as heme by-products, catalase, and triglycerides. In some embodiments, the pigment material can include a metallic material or metal. In some embodiments, the metal is titanium, such as, but not limited to, titanium dioxide (TiO). The titanium dioxide can be an anatase titanium dioxide pigment, which has high whiteness and a blue hue. In some embodiments, the pigment material is Hombitan LC-S, Huntsman TiO, or Kemiera 300. In other embodiments, the titanium dioxide can be other crystalline forms, such as, but not limited to, rutile, brookite, akaogiite, and combinations thereof, or combinations with anatase.

[0082] In some embodiments, titanium dioxide and hardened gel work together to produce a sieve that allows small molecular weight species (such as Fru-α-ValHis) to easily pass through the layer while excluding larger molecular weight proteins (hemoglobin, catalase, proteases). In one embodiment, a sieve is produced that allows Fru-α-ValHis to pass through.

[0083] In some embodiments, this exclusion of larger molecular weight proteins may be an essential feature of the masking layer because hemoglobin causes optical interference in HbA1c measurements at 670 nm, catalase consumes peroxides required for dye oxidation, and / or proteases digest registered enzymes present in the gel layer (fructosyl peptide oxidase, horseradish peroxidase).

[0084] In some embodiments, titanium dioxide can provide a uniform reflective surface used to reflect 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 the light contacts or otherwise interacts with the sample, one or more sensors can be used to read the amount of light at one or more wavelengths. The sensor can be a photomultiplier tube, a contact image sensor, an image capture sensor matrix, or a combination thereof.

[0085] Buffering the masking layer serves to maintain the masking layer at a desired pH, which 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.

[0086] In one embodiment, the at least one masking layer buffer can include a first buffer and a second buffer, each of which can be an acid or base salt, as appropriate. In one embodiment, the first buffer is 3-(N-morpholino)propanesulfonic acid (MOPS). In one embodiment, the second buffer is β,β-dihydroxyl-1,4-piperazinedipropanesulfonic acid disodium salt (POPSO).

[0087] The dispersant can be sodium polymethacrylate. This agent can be effective in rapidly dispersing pigments. In one embodiment, the dispersant is Daxad 30S.

[0088] The masking layer surfactant can act as a coating aid. The masking layer surfactant can be an anionic surfactant such as a polyethersulfonate. In one embodiment, the masking layer surfactant is TRITON X200E.

[0089] The hardener can function to crosslink the gel in the gel layer and / or promote cohesion of the layer, hi some embodiments, the hardener is bis(vinylsulfonylmethyl) (BVSM).

[0090] In some embodiments, the solvent / diluent for the masking layer is water.

[0091] The masking layer 104 can separate functional areas of the slide. For example, the masking layer 104 can separate the spread layer 108 from the gel layer 102. This allows the red blood cell lysis, hemoglobin denaturation / digestion, and Fru-α-ValHis dipeptide release that occur in the spread layer to be separated from the Fru-α-ValHis dipeptide deglycosylation and HRP / dye reaction to generate a colorimetric signal that occur in the gel layer 102.

[0092] In some embodiments, the masking layer is not present, for example, when forming slides requiring measurement of Hb at 540 nm, titanium dioxide is not present as it would interfere with the ability to read hemoglobin.

[0093] However, in other embodiments where measurement of Hb at 540 nm is desired, the masking layer 104 may still contain titanium dioxide. In some embodiments, a reflectance signal for Fru-α-ValHis at 670 nm can be generated in the gel layer, and a reflectance signal for Hb at 540 nm that has not passed through the masking layer can be generated above the masking layer. In such embodiments, two separate reflectance signals are measured: one from above to measure Hb at 540 nm and one from below to measure Fru-α-ValHis at 670 nm. These values ​​can be used to determine the derived %HbA1c.

[0094] The gel layer 102 can include a gel, a buffer, at least one surfactant, a coupler solvent, a reducing agent, a detection agent, a cofactor, an amplification substance or catalyst, an oxidase, a hardener, and a solvent / diluent.

[0095] In some embodiments, the gel layer is a gel or gelatin. The gel can promote layer cohesion, promote capillary forces upon rewetting, and / or provide a size exclusion mechanism. In some embodiments, the size exclusion mechanism can exclude high molecular weight interferents (upon crosslinking / hardening). In some embodiments, the gel is a crosslinked gel. Crosslinking can improve layer integrity and reduce pore size to filter out additional interferents.

[0096] In one embodiment, the gel is Gel-32 TCGIII DI Geratin. The gel can be porous.

[0097] In some embodiments, the gel can function as a size exclusion mechanism.

[0098] The gel layer buffer can function 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 appropriate. In one embodiment, the buffer is 3-(N-morpholino)propanesulfonic acid (MOPS).

[0099] The gel layer 102 can include one or more surfactants. A first surfactant can function as a coating aid. In some embodiments, the first surfactant in the gel layer is an octylphenol ethoxylate, such as TRITON X-100.

[0100] A second surfactant can be used to disperse the dye. In one embodiment, the second surfactant is a sodium alkylated naphthalene sulfonate such as Alkanol XC.

[0101] The dye layer coupler solvent can be 2,4-di-n-pentylphenol (KS-52) and / or 2,4-di-tert-pentylphenol (KS-41).

[0102] A reducing agent can be added to prevent accidental oxidation of the dye. In one embodiment, the reducing agent can be 5,5-dimethyl-1,3-cyclohexanedione (Dimedone).

[0103] The detecting agent may be a dye. The detecting agent may be used to generate a colorimetric signal. Virtually any dye that provides a detectable signal may be used. The dye may be N-carboxymethylaminocarbonyl. Ru- The dye can be 4,4'-bis(dimethylamino)-diphenylamine sodium (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), 2-(3,5-dimethoxy-4-hydroxyphenol)-4,5-bis-(4-dimethylaminophenyl)imidazole, or a combination thereof. In one embodiment, the dye is 2-(3,5-dimethoxy-4-hydroxyphenol)-4,5-bis-(4-dimethylaminophenyl)imidazole.

[0104] The oxidase can be an oxidase that generates peroxide from Fru-α-ValHis. The oxidase can deglycosylate Fru-α-ValHis to generate peroxide. In one embodiment, the oxidase can be a fructosyl peptide oxidase.

[0105] In some embodiments, the gel layer 102 can include an oxidase reaction cofactor. The cofactor can be a non-protein chemical compound that supports oxidase activity. In other embodiments, the cofactor can be flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and / or coenzyme A (CoA). In one embodiment, the cofactor can be flavin adenine dinucleotide (FAD).

[0106] The amplification agent or catalyst can be any molecule that amplifies the reaction of the colorimetric dye. Peroxide generated from the Fru-α-ValHis oxidase reaction can interact with the dye, whose signal is amplified by the amplification agent. In some embodiments, the amplification agent is a peroxidase, such as, but not limited to, horseradish peroxidase (POD).

[0107] The hardener can function to crosslink the gel in the gel layer and / or promote cohesion of the layer, hi some embodiments, the hardener is bis(vinylsulfonylmethyl) (BVSM).

[0108] In some embodiments, the solvent / diluent for the gel layer is water.

[0109] In some embodiments, in the gel layer 102, Fru-α-ValHis is deglycosylated by fructosyl peptide oxidase. Fructosyl oxidase is specific for both Fru-α-ValHis and Fru-α-Val. However, proteolysis of the β-chain of hemoglobin does not produce Fru-α-Val. The specificity of fructosyl oxidase also prevents interference of the assay with other glycated proteins, such as albumin.

[0110] The desaccharification reaction recycles the FAD cofactor and produces peroxide. Horseradish peroxidase and peroxide oxidize the dye to a colored product that absorbs light at 670 nm.

[0111] In some embodiments, the hardened gel of gel layer 102 is an additional protective layer to exclude larger molecular weight proteins (hemoglobin, catalase, proteases). Cross-linking the gel reduces the pore size of the layer and also helps prevent dye particles from the gel layer from mixing with the masking layer (e.g., melt) components during coating.

[0112] In some embodiments, hardening of the gel layer 102 can increase the signal for a microslide. 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%.

[0113] In some embodiments, the interference inhibitor can be ascorbic acid oxidase. This oxidase can react with ascorbic acid (vitamin C) in the sample and prevent it from interfering with the measurement. In some embodiments, ascorbic acid in the sample can react with dyes in the gel layer, reducing them and reducing their color. This color reduction can result in negative %A1c prediction bias, or simply falsely low %A1c results. Inclusion of ascorbic acid oxidase can remove ascorbic acid from the sample, preventing negative prediction bias.

[0114] In certain embodiments, samples may contain megadoses of ascorbic acid from patients using large amounts of vitamins to alleviate or relieve certain symptoms. Absence of an anti-interference agent, such as ascorbic acid oxidase, can lead to inaccurate results. Thus, in some embodiments, the slides described herein include an anti-interference agent in the gel layer or any other suitable layer of the slide.

[0115] In some embodiments, the dyes on the microslides are analyzed using a detection paradigm. The detection paradigm can be one or more reflectance measurements. In one embodiment, reflectance measurements are used to detect absorbance by the dyes described herein.

[0116] In one embodiment, light of a specific wavelength is directed onto the detection area 128 and the reflected light or reflectance density at the specific wavelength is measured. Reflectance density (DR) is determined from the reflectance. Reflectance density is equal to the reciprocal Log of the reflectance.

[0117] In some embodiments, the light is reflected off a titanium dioxide layer within the microslide. In some embodiments, the specific wavelength of light can be 540 nm, 670 nm, or both. In some embodiments, wavelengths around these values ​​or a range of wavelengths including these values ​​can be used, depending on the signal strength and / or potential interferents that absorb light in the same spectrum.

[0118] In some embodiments, wavelengths in the Soret and Q band regions can be used, including, but not limited to, wavelengths of about 540 nm, such as 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 range of 530 nm to 540 nm, the range of 539 nm to 541 nm, the range of 538 nm to 542 nm, the range of 537 nm to 543 nm, the range of 536 nm to 544 nm, the range of 535 nm to 545 nm, the range of 540 nm to 545 nm, the range of 535 nm to 540 nm, the range of 535 nm to 575 nm, the range of 530 nm to 575 nm, the range of 540 nm to 575 nm, the range of 550 nm to 575 nm, or the range of 560 nm to 575 nm can be used.

[0119] Similarly, in some embodiments, a wavelength of about 670 nm can be used, including, but not limited to, wavelengths of about 665 nm, about 666 nm, about 667 nm, about 668 nm, about 669 nm, about 671 nm, about 672 nm, about 673 nm, about 674 nm, or about 675 nm. In other embodiments, a wavelength range can be used, including, but not limited to, wavelengths in the range of 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, or 665 nm to 670 nm.

[0120] In one embodiment, the reflectance density is read by an automated analyzer, such as, but not limited to, a VITROS analyzer. The endpoint reflectance density or reflectance may be quantified from the dye produced by this oxidation reaction.

[0121] Assay times may vary depending on the analytical protocol or analytical instrument used. However, generally, the time from sample application through the enzyme cascade to quantification of reflectance concentration is about 5 to about 10 minutes, about 4 to about 6 minutes, about 3 to about 7 minutes, about 2 to about 8 minutes, less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, or less than about 5 minutes. In one embodiment, a typical assay time is about 5 minutes at 37°C on a VITROS analyzer.

[0122] The assay can be performed on a VITROS analyzer at 37°C, although other temperatures are also possible. For example, in some embodiments, the assay can be performed at room temperature, or at temperatures above or below 37°C.

[0123] In some embodiments, components of the enzyme cascade, such as proteases, fructosyl oxidase, peroxidase, dyes, and / or FAD, may be omitted from the gel layer, as they are not necessary for reading the hemoglobin signal at 540 nm.

[0124] In some embodiments, a single microslide can be used to measure only %A1c. In other embodiments, separate microslides can be used to measure Hb and HbA1c separately to obtain a derived %A1c result. Alternatively, all components necessary to determine Hb and HbA1c concentrations can be incorporated into a single microslide to obtain a derived %A1c result.

[0125] The devices, systems, and methods described herein can use whole blood patient samples without dilution or pretreatment. In some embodiments, the whole blood is lysed. Using whole blood can save time and resources compared to testing systems that require processed blood.

[0126] In some embodiments, the devices, systems, and methods described herein can measure HbA1c levels using small amounts of blood. In clinical and diagnostic settings, blood sample volume can be important, especially when large test panels are performed.

[0127] The small amount of blood can be about 1 μL to about 10 μL, about 2 μL to about 8 μL, about 4 μL to about 6 μL, about 4 μL to about 5 μL, about 4 μL to about 10 μL, about 2 μL to about 5 μL, less than about 10 μL, less than about 8 μL, less than about 6 μL, or less than about 5 μL.

[0128] In some embodiments, the devices, systems, and methods described herein can measure HbA1c levels in a short time compared to conventional methods, which can be referred to as a rapid assay time. In clinical and diagnostic settings, measurement time can be important when considering time and equipment throughput costs.

[0129] The rapid assay time can be from about 1 minute to about 10 minutes, from about 2 minutes to about 8 minutes, from about 3 minutes to about 7 minutes, from about 4 minutes to about 7 minutes, from about 4 minutes to about 8 minutes, from about 5 minutes to about 7 minutes, from about 6 minutes to about 8 minutes, from about 5 minutes to about 8 minutes, less than about 10 minutes, less than about 8 minutes, less than about 7 minutes, or less than about 6 minutes.

[0130] Rapid assay times allow high-throughput systems utilizing the assays described herein to perform more assays per hour than conventional assays, thereby generating more profit per hour. In some embodiments, a high-throughput system is capable of performing between about 300 tests / hour and about 400 tests / hour, between about 350 tests / hour and about 400 tests / hour, between about 350 tests / hour and about 450 tests / hour, between about 300 tests / hour and about 500 tests / hour, between about 300 tests / hour and about 600 tests / hour, at least about 300 tests / hour, at least about 350 tests / hour, at least about 375 tests / hour, or at least about 400 tests / hour.

[0131] The devices, systems, and methods described herein can ensure assay specificity through proteolysis of the N-terminal β-chain of hemoglobin to generate a substrate (Fru-α-ValHis) and deglycosylation by a specific fructosyl peptide oxidase.

[0132] In some embodiments, the devices, systems, and methods are not affected by hemoglobin structural variant (HbS, HbC) interference, which some commercially available assays suffer from because they are antibody-based methods.

[0133] Methods of using the microslides described herein are also described.

[0134] In one embodiment, a single-slide method for detecting hemoglobin and glycated hemoglobin is described. The method includes contacting a spread layer of a microslide, such as the slides 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 and modifies the glycated hemoglobin, and a protease releases fructosyl peptides from the modified glycated hemoglobin. The fructosyl peptides then reach the gel layer and contact fructosyl oxidase, which releases peroxide. The peroxidase and peroxide then contact a detection agent, which releases and / or generates a detectable signal.

[0135] In some embodiments, the anti-interference agent reacts with any ascorbic acid in the blood sample to prevent sample bias, hi some embodiments, the anti-interference agent is ascorbic acid oxidase.

[0136] The amount of hemoglobin is measured from a blood sample. The measurement includes reading a reflectance density of the sample using light at a first wavelength. The amount of glycated hemoglobin from the blood sample is also measured. The measurement of glycated hemoglobin includes detecting a reflectance density of a detectable signal from the sample with light at a second wavelength. In some embodiments, the second wavelength of light is different from the first wavelength of light. In one embodiment, the first wavelength of light is 540 nm and the second wavelength of light is 670 nm.

[0137] In some embodiments, light at a first wavelength can be measured relatively early in the analysis, and light at a second wavelength can be measured later in the analysis after a lag time. The lag time can be about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 30 seconds to about 1 minute, about 40 seconds to about 1 minute, about 30 seconds to about 2 minutes, about 30 seconds to about 3 minutes, or about 30 seconds to about 4 minutes. This lag time allows sufficient time for the reaction cascade to occur.

[0138] In another embodiment, a single-slide method for directly detecting glycated hemoglobin is described. The method includes contacting a first film layer of a microslide 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 and denatures the glycated hemoglobin, and a protease releases fructosyl peptides from the denatured glycated hemoglobin. The fructosyl peptides then traverse the second film layer, reach the third film layer, and contact fructosyl oxidase, releasing peroxide. The peroxidase and peroxide react with a detection agent to generate a detectable signal.

[0139] In some embodiments, the anti-interference agent reacts with any ascorbic acid in the blood sample to prevent sample bias, hi some embodiments, the anti-interference agent is ascorbic acid oxidase.

[0140] The amount of glycated hemoglobin is then measured from the blood sample. In such embodiments, no measurement of non-glycated hemoglobin is performed. Measuring glycated hemoglobin includes detecting the reflectance concentration of a detectable signal in the sample. Measuring glycated hemoglobin includes detecting the reflectance concentration of a detectable signal from the sample at a wavelength of light. In some embodiments, the wavelength of light is 670 nm.

[0141] In some embodiments, the microslides described herein can have a lower unit manufacturing cost when compared to conventional HbA1c measurement assays. The microslides can reduce manufacturing costs by about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%.

[0142] The microslides described herein can be manufactured by coating successive film layers onto a transparent support. Thus, a microslide can be manufactured by applying a gel layer coating onto the support (114), followed by a masking layer on the gel layer, an adhesive layer on the masking layer, and a spreading layer on the adhesive layer.

[0143] In another embodiment, once the coating has been formed by successive thin film layer deposition, it is notched to the appropriate width. The notched coating can then be assembled into a finished microslide by carving the notches into individual slide-sized chips, which can then be attached to top and bottom slide mounts along with spacer webs. This process can be performed on a slide assembly machine (SAM).

[0144] Individual slides can be packaged in carts for use with mainframe analyzers. Carts can contain any number of slides appropriate for the analyzer. In some embodiments, carts can hold 50 microslides, 100 microslides, 200 microslides, at least 10 microslides, at least 15 microslides, at least 20 microslides, at least 50 microslides, or at least 100 microslides. In other embodiments, carts can hold 18 microslides, 50 microslides, or 60 microslides.

[0145] In some embodiments, the components of the spread layer can be applied using an inkjet deposition process. The components of the spread layer applied by the inkjet deposition process can include a protease, sodium nitrite, and / or calcium chloride.

[0146] In some embodiments, the microslide can include a spacer web 130 between the support layer 114 and the lower slide mount 112. The spacer can prevent damage to the microslide during assembly, particularly during welding of the upper and lower slide mounts.

[0147] In some embodiments, the microslide has a thickness of about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, or about 600 μm, including the spreading layer, adhesive layer, masking layer, gel layer, optional spacer web, and support layer.

[0148] Example 1 (Using a TiO2 masking layer to reduce hemoglobin optical interference) Six microslides are prepared: four with TiO2 in the masking layer and two without. A blood sample is placed on each slide on the spread layer.

[0149] Figure 2 provides a pictorial representation of the effect of the TiO2 masking layer. Slides 1 and 3 show the spot side of the TiO2 masking layer slide. Hemoglobin is visible on the spot side of the slide. Slides 2 and 4 show the read side of the TiO2 masking layer slide. Hemoglobin has been excluded from the gel layer side (read side) of the slide.

[0150] In contrast, slides 5 and 6 represent the spot side (slide 5) and the read side (slide 6), respectively, of a microslide that does not contain TiO2 in the masking layer. Hemoglobin can be easily identified on slide 6 (read side).

[0151] Thus, the inclusion of TiO2 in the masking layer can prevent significant amounts of hemoglobin from penetrating the gel layer.

[0152] Figures 3A and 3B and Figures 4A and 4B are kinetic plots for a 10-level hemolysis series ranging in hemoglobin concentration from about 6 g / dL to about 20 g / dL for the "control coating" (no TiO2 masking layer) and the TiO2 masking layer.

[0153] The kinetics were recorded over a 5-minute period at wavelengths of 540 nm and 670 nm. Note the different scales on the plots. For the control coating, the hemoglobin signal at 540 nm shows a typical kinetic profile. The response at 540 nm of the TiO2 masking layer slide indicates that the 540 nm signal was masked by excluding hemoglobin from the gel layer of the coating.

[0154] Similarly, the hemoglobin signal at 670 nm for the control coating exhibits a typical kinetic profile. This data indicates that the hemoglobin signal at 670 nm varies depending on the hemoglobin concentration being evaluated. This signal may lead to optical interference, requiring a correction algorithm, for the 670 nm dye readout of the HbA1c assay. The TiO2 contained in the masking layer significantly reduces the hemoglobin signal at 670 nm, preventing optical interference with the HbA1c colorimetric assay (readout at 670 nm).

[0155] Figures 5 and 6 show dose-response plots of pure Fru-α-ValHis dipeptide in solutions with increasing hemoglobin concentrations, compared with masking layer slides containing TiO2 and without TiO2. In Figure 5, the solution is run over a slide without TiO2. The data show that with increasing hemoglobin concentration, the background signal at 670 nm (0.0 mM Fru-α-ValHis) increases, with a loss in the delta signal across the Fru-α-ValHis levels tested. In Figure 6, the solution is run over a masking layer slide containing TiO2. The data show that with increasing hemoglobin concentration, the background signal at 670 nm (0.0 mM Fru-α-ValHis) remains constant. The optical interference of hemoglobin at 670 nm is reduced and / or eliminated.

[0156] Example 2 (HbA1c microslide test) Evaluating HbA1c microslide data for model %A1c fluid and whole blood patient samples. Figures 7A-D show reflectance density (DR) kinetic data at 670 nm for comparison of signal generation for model %A1c fluid and %A1c patient samples.

[0157] The A1c model fluid and %A1c patient samples have comparable %A1c. All HbA1c microslides are hopper coated, except for the sodium nitrite and protease, which are applied by an inkjet deposition process.

[0158] Furthermore, coating with a high concentration (coverage) of a denaturing surfactant (N-lauroyl sarcosine, NLS) improves the dynamic response of the %A1c model fluid and the %A1c patient sample. As can be seen from Figures 7A-D, the %A1c patient sample has a kinetic profile similar to the %A1c model fluid. Figures 7A-D also demonstrate good discrimination between the evaluated %A1c levels.

[0159] 8A and 8B show DR kinetic data at 670 nm for a comparison of signal generation of a model %A1c fluid and a %A1c patient sample, generated with an x-hopper coating incorporating all components to perform the enzyme cascade described herein.

[0160] The %A1c model fluid and the %A1c patient samples have comparable %A1c. As can be seen from Figures 8A and 8B, the %A1c patient samples have a similar kinetic profile to the %A1c model fluid. Figures 8A and 8B also demonstrate good discrimination between the assessed %A1c levels.

[0161] Figure 9 shows DR dose-response data at 670 nm for the %A1c model fluid and whole blood %A1c patient sample. This data was generated using the same coating as the data shown in Figures 8A and 8B. This data clearly shows that the whole blood patient sample gave the same response curve as the %A1c model fluid made from purified glycated hemoglobin. This demonstrates that the microslides described herein can lyse red blood cells and denature and digest glycated hemoglobin to produce a substrate that leads to a colorimetric signal.

[0162] Figure 10 shows a correlation plot between patient sample predicted %A1c results for HbA1c microslides and assigned HPLS %A1c values. A linear calibration model based on the microchip %A1c reference and HbA1c microslide reflectance concentration at 670 nm is used to predict each of six replicate microslides for each patient sample. The average microslide %A1c prediction is compared to the BioRad Variant high-performance liquid chromatography (HPLC) %A1c results. As evidenced in Figure 10, the microslide %A1c assay has a very strong correlation with the HPLC %A1c assay.

[0163] Based on the data obtained and discussed in this example, direct measurement of %A1c is feasible using the microslides described herein.

[0164] Example 3 (%A1c derived using measured values ​​of HbA1c and hemoglobin components) Here, the HbA1c microslide described herein is used in combination with a hemoglobin microslide to generate derived %A1c results for patient samples.

[0165] Here, the HbA1c microslide measures the component glycated hemoglobin, and the separate hemoglobin microslide measures the total hemoglobin component, and these two results are used to calculate the derived %A1c test result.

[0166] Figure 11 shows a dose-response plot of the hemoglobin component of the %A1c patient samples used in the previous section. Reflectance density at 540 nm is plotted against the Vitros Microtip hemoglobin concentration results (g / dL).

[0167] Figure 12 shows a correlation plot of predicted Microslide hemoglobin component concentrations for patient samples versus MicroTip reference hemoglobin values. A linear calibration model based on MicroTip hemoglobin reference values ​​and hemoglobin Microslide reflectance at 540 nm is used to predict each of the six hemoglobin Microslide replicates for each patient sample (Figure 11). The mean values ​​of the predicted Microslide hemoglobin component values ​​are compared to the MicroTip hemoglobin reference values. The Microslide hemoglobin component assay correlates well with the MicroTip hemoglobin assay.

[0168] Figure 13 shows the dose-response plot of HbA1c component for the %A1c patient samples used in Example 2. Reflectance density at 670 nm is plotted against MicroTip HbA1c component concentration results (g / dL).

[0169] Figure 14 shows a correlation plot between the predicted Microslide HbA1c component concentrations of patient samples and the assigned MicroTip reference HbA1c component values. A linear calibration model based on the MicroTip HbA1c component reference values ​​and the HbA1c Microslide reflectance at 670 nm is used to predict each of the six HbA1c component replicate Microslides for each patient sample (Figure 13). The average of the predicted Microslide HbA1c component values ​​is compared to the MicroTip HbA1c component reference values. The Microslide HbA1c component assay has good correlation with the MicroTip HbA1c component assay.

[0170] Figure 15 shows a correlation plot of predicted MicroSlide derived %A1c and BioRad Variant HPLC reference %A1c values ​​for patient samples. MicroSlide %A1c values ​​are obtained by using the hemoglobin component results (g / dL) and the HbA1c component results (g / dL) to generate the derived %A1cwp using the National Glycohemoglobin Standardization Program "Master Formula" (VITROS HbA1c MicroTip Assay Instructions for Use, Pub. J55871_EN, Version 2.0).

number

[0171] The mean microslide %A1c values ​​are compared to the HPLC %A1c reference values. The microslide derived %A1c assay correlates well with the HPLC %A1c assay.

[0172] The data in Example 3 demonstrate the feasibility of using the hemoglobin microslide assay described herein in combination with the HbA1c microslide assay to generate derived %A1c results.

[0173] Example 4 (Derived %A1c using determination of HbA1c and hemoglobin components) Here, a single microslide test element is used to generate both hemoglobin spectral results and HbA1c enzymatic results to generate derived %A1c results for patient samples. This is called a "dual-assay" microslide test element. The microslides used do not contain titanium dioxide in the masking layer, as this would inhibit the ability to read the 540 nm hemoglobin signal. The protease and sodium nitrite can be incorporated into the microslide by inkjet deposition or x-hopper coating processes.

[0174] Figures 16 and 17 show the HbA1c component dose-response data (670 nm) and hemoglobin component dose-response (540 nm) of the dual assay microslide test element. The reflectance density (DR) dose-response data demonstrate that the dual assay microslide test element is able to register the BBI HbA1c liquid series in a dose-dependent manner at 670 nm, indicating that the enzyme cascade is functioning. The microslide test element also measures the hemoglobin spectrum read at 540 nm in a dose-dependent manner. As described in Example 3, derived %A1c measurements are feasible using the "dual assay" microslide test element.

[0175] Example 5 (Direct measurement of %A1c) A whole blood sample is applied to a microslide as described herein. The enzyme cascade described herein produces an oxidation dye that absorbs light at 670 nm, and the microslide uses a masking layer to filter out non-glycated hemoglobin. The light is reflected off titanium dioxide in the masking layer, and the reflectance density is read at 670 nm. %A1c is calculated directly from the reflectance density using a %A1c calibration curve.

[0176] Example 6 (%A1c derived using one sample) A whole blood sample is applied to a microslide described herein that does not contain titanium dioxide in the masking layer. The enzyme cascade described herein produces an oxidation dye that absorbs light at 670 nm and allows measurement of non-glycated hemoglobin at 540 nm. The derived %A1c can be calculated from the measurements at 540 nm and 670 nm.

[0177] Example 7 (%A1c derived using two samples on a single microslide) Two whole blood samples are applied separately to two areas of a microslide. One area contains titanium dioxide in a masking layer, and the enzyme cascade described herein generates an oxidation dye that absorbs light at 670 nm. The other area does not contain titanium dioxide, allowing direct measurement of non-glycated hemoglobin at 540 nm. The derived %A1c can be calculated from the measurements at 540 nm and 670 nm.

[0178] Example 8 (%A1c derived using two measurements on a single slide) A whole blood sample is applied to a microslide described herein. The enzyme cascade described herein produces an oxidation dye that absorbs light at 670 nm, and the microslide's masking layer eliminates the signal of non-glycated hemoglobin. Light is reflected from beneath the masking layer using titanium dioxide, and the reflectance density is read at 670 nm. Light is also reflected from above the masking layer using titanium dioxide and VtE beads in the spreading layer, and the reflectance density of hemoglobin is read at 540 nm. The measurements at 540 nm and 670 nm allow the calculation of a derived %A1c.

[0179] Example 9 (Reducing ascorbic acid interference) Two samples are taken from a patient who has been megadosed with ascorbic acid for its anti-cancer effects: the first sample is measured on a slide containing ascorbic acid oxidase in its gel layer, and the second sample is measured on a slide without ascorbic acid oxidase.

[0180] The results from the first sample show a higher %A1c value than the second sample.

[0181] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed by applying ordinary rounding factors in light of, at least, the number of reported significant digits. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0182] As used in the context of describing the present invention (particularly in the context of the claims below), the terms "a," "an," "the," and similar modifiers should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referencing each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if individually referenced herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to facilitate a better understanding of the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0183] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually, or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to include the modified group and, accordingly, conforms to all Markush-style group descriptions used in the appended claims.

[0184] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will occur to those skilled in the art, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0185] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely as shown and described.

[0186] [Note] [Appendix 1] a first film layer comprising a cross-linked gel, the cross-linked gel comprising a detection agent, a fructosyl oxidase, an anti-interference agent, and a peroxidase; a second film layer comprising the first gel; a third film layer comprising a lysis agent, a denaturant, and a protease; A slide comprising a stack of film layers comprising, from bottom to top:

[0187] [Appendix 2] The dissolving agent is a detergent. 2. The slide of claim 1.

[0188] [Appendix 3] The detergent is selected from the group consisting of octylphenol ethoxylate (TRITON X-100), TWEEN (TWEEN 20), sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ether (POE), and NONIDET P-40 (NP-40); 3. The slide of claim 2.

[0189] [Appendix 4] The detergent is octylphenol ethoxylate. 4. The slide of claim 3.

[0190] [Appendix 5] The denaturing agent is a surfactant. 2. The slide of claim 1.

[0191] [Appendix 6] The denaturant is one or more of sodium nitrite or N-lauroyl sarcosine (NLS). 6. The slide of claim 5.

[0192] [Appendix 7] The protease is a metalloprotease. 2. The slide of claim 1.

[0193] [Appendix 8] the third film layer further comprises calcium; 8. The slide of claim 7.

[0194] [Appendix 9] The protease is a neutral protease. 9. The slide of claim 8.

[0195] [Appendix 10] The protease is an endoprotease or an exoprotease. 2. The slide of claim 1.

[0196] [Appendix 11] the protease is selected from the group consisting of proteinase K, pronase E, protease XVII, protease XXI, aminopeptidase, carboxypeptidase, thermolysin, and subtilisin; 11. The slide of claim 10.

[0197] [Appendix 12] The peroxidase is horseradish peroxidase. 2. The slide of claim 1.

[0198] [Appendix 13] The detection agent is N-carboxymethylaminocarbonyl Ru- selected from the group consisting of 4,4'-bis(dimethylamino)-diphenylamine sodium (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; 2. The slide of claim 1.

[0199] [Appendix 14] the second film layer further comprises a reflective material portion; 2. The slide of claim 1.

[0200] [Appendix 15] The reflective material portion includes a metal. 15. The slide of claim 14.

[0201] [Appendix 16] The metal is titanium. 16. The slide of claim 15.

[0202] [Appendix 17] the third film layer further comprises a layer comprising particles having a diameter of about 25 μm; 2. The slide of claim 1.

[0203] [Appendix 18] the third film layer further comprises an oxidase cofactor and a surfactant; 2. The slide of claim 1.

[0204] [Appendix 19] the oxidase cofactor is flavin adenine dinucleotide (FAD); 19. The slide of claim 18.

[0205] [Appendix 20] the fructosyl oxidase is specific for a Fru-α-ValHis peptide or a Fru-α-Val amino acid; 19. The slide of claim 18.

[0206] [Appendix 21] the first film layer is in direct contact with the lower surface of the second film layer; 2. The slide of claim 1.

[0207] [Appendix 22] the second film layer directly contacts the lower surface of the third film layer; 22. The slide of claim 21.

[0208] [Appendix 23] The anti-interference agent is ascorbic acid oxidase. 2. The slide of claim 1.

[0209] [Appendix 24] a) providing a slide according to Appendix 1; b) contacting the third film layer of the slide with an unlysed blood sample containing red blood cells, wherein the lysing agent releases glycated hemoglobin from the red blood cells, the denaturing agent contacts and denatures the glycated hemoglobin, the protease releases fructosyl peptides from the denatured glycated hemoglobin, the fructosyl peptides reach the first film layer and contact the fructosyl oxidase to release peroxide, and the peroxidase and the peroxide contact the detecting agent to emit a detectable signal; c) measuring the amount of hemoglobin from the blood sample, the step of measuring the amount of hemoglobin comprising reading a reflectance density of the sample from the slide at a first wavelength of light; and d) measuring the amount of glycated hemoglobin from the blood sample, wherein measuring the amount of glycated hemoglobin comprises detecting a reflectance density of the detectable signal from the sample at a second wavelength of light, wherein the second wavelength of light is different from the first wavelength of light; 1. A single-slide method for detecting hemoglobin and glycated hemoglobin, comprising:

[0210] [Appendix 25] the first wavelength of light is 540 nm and the second wavelength of light is 670 nm; 25. The method of claim 24.

[0211] [Appendix 26] the dissolving agent is a detergent selected from the group consisting of octylphenol ethoxylate (TRITON X-100), TWEEN (TWEEN 20), sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ether (POE), and NONIDET P-40 (NP-40); 25. The method of claim 24.

[0212] [Appendix 27] The detergent is octylphenol ethoxylate. 27. The method of claim 26,

[0213] [Appendix 28] The denaturant is one or more of sodium nitrite or N-lauroyl sarcosine (NLS). 25. The method of claim 24.

[0214] [Appendix 29] The protease is a metalloprotease, an endoprotease, or an exoprotease. 25. The method of claim 24.

[0215] [Appendix 30] The metalloprotease is a neutral protease. 30. The method of claim 29,

[0216] [Appendix 31] contacting the fructosyl peptide with an oxidase cofactor that is flavin adenine dinucleotide (FAD); 25. The method of claim 24.

[0217] [Appendix 32] The peroxidase is horseradish peroxidase. 25. The method of claim 24.

[0218] [Appendix 33] the disclosing agent is a leuco dye; 25. The method of claim 24.

[0219] [Appendix 34] a) providing a slide according to claim 14; b) contacting the third film layer of the slide with a blood sample containing red blood cells, wherein the lysing agent releases glycated hemoglobin from the red blood cells, the denaturing agent contacts and denatures the glycated hemoglobin, the protease releases fructosyl peptides from the denatured glycated hemoglobin, the fructosyl peptides cross the second film layer, the fructosyl peptides reach the first film layer and contact the fructosyl oxidase to release peroxide, and the peroxidase and the peroxide contact the detecting agent to emit a detectable signal; and c) measuring the amount of glycated hemoglobin from the blood sample, wherein the step of measuring the amount of glycated hemoglobin comprises detecting a reflectance concentration of the detectable signal in the sample; A single-slide method for directly detecting glycated hemoglobin, comprising:

[0220] [Appendix 35] The reflective material portion includes a metal. 35. The method of claim 34.

[0221] [Appendix 36] The fructosyl peptide crosses the cross-linked gel. 35. The method of claim 34.

[0222] [Appendix 37] The protease is a metalloprotease, an endoprotease, or an exoprotease. 35. The method of claim 34.

[0223] [Appendix 38] The metalloprotease is a neutral protease. 38. The method of claim 37,

[0224] [Appendix 39] contacting the fructosyl peptide with an oxidase cofactor that is flavin adenine dinucleotide (FAD); 35. The method of claim 34.

[0225] [Appendix 40] The peroxidase is horseradish peroxidase. 35. The method of claim 34.

[0226] [Appendix 41] the disclosing agent is a leuco dye; 35. The method of claim 34.

Claims

1. a first film layer comprising a cross-linked gel, the cross-linked gel comprising a detection agent, a fructosyl oxidase, an anti-interference agent, and a peroxidase; a second film layer comprising a gel; a third film layer comprising particles, a lysing agent, a denaturing agent, and a protease; 1. A slide comprising a stack of film layers comprising, from bottom to top, the second film layer further comprises a reflective material portion; A slide characterized by:

2. The dissolving agent is a detergent.

2. The slide of claim 1.

3. the detergent is selected from the group consisting of octylphenol ethoxylate (TRITON® X-100), polyethylene glycol sorbitan monolaurate (TWEEN® 20), sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ether (POE), and octylphenoxypolyethoxyethanol (NONIDET® P-40); 3. The slide of claim 2.

4. The detergent is octylphenol ethoxylate.

4. The slide of claim 3.

5. the denaturing agent is one or more of a surfactant, sodium nitrite, or N-lauroyl sarcosine (NLS); 2. The slide of claim 1.

6. The protease is a metalloprotease.

2. The slide of claim 1.

7. a first film layer comprising a cross-linked gel, the cross-linked gel comprising a detection agent, a fructosyl oxidase, an anti-interference agent, and a peroxidase; a second film layer comprising a gel; a third film layer comprising a lysis agent, a denaturant, and a protease; 1. A slide comprising a stack of film layers comprising, from bottom to top, the protease is a metalloprotease; the third film layer further comprises calcium; A slide characterized by:

8. The protease is a neutral protease.

8. The slide of claim 7.

9. The protease is an endoprotease or an exoprotease.

2. The slide of claim 1.

10. the protease is selected from the group consisting of proteinase K, pronase E, protease XVII, protease XXI, aminopeptidase, carboxypeptidase, thermolysin, and subtilisin; 10. The slide of claim 9.

11. The peroxidase is horseradish peroxidase.

2. The slide of claim 1.

12. the disclosing agent is selected from the group consisting of N-carboxymethylaminocarbonyl-4,4'-bis(dimethylamino)-diphenylamine sodium (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; 2. The slide of claim 1.

13. The reflective material portion includes a metal.

2. The slide of claim 1.

14. The metal is titanium.

14. The slide of claim 13.

15. a first film layer comprising a cross-linked gel, the cross-linked gel comprising a detection agent, a fructosyl oxidase, an anti-interference agent, and a peroxidase; a second film layer comprising a gel; a third film layer comprising particles, a lysing agent, a denaturing agent, and a protease; 1. A slide comprising a stack of film layers comprising, from bottom to top, the particles of the third film layer have a diameter of 25 μm; A slide characterized by:

16. the third film layer further comprises an oxidase cofactor and a surfactant; 2. The slide of claim 1.

17. The oxidase cofactor is flavin adenine dinucleotide (FAD).

17. The slide of claim 16.

18. the fructosyl oxidase is specific for a Fru-α-ValHis peptide or a Fru-α-Val amino acid; 17. The slide of claim 16.

19. the first film layer is in direct contact with the underside of the second film layer; 2. The slide of claim 1.

20. the second film layer directly contacts the lower surface of the third film layer; 20. The slide of claim 19.

21. The anti-interference agent is ascorbic acid oxidase.

2. The slide of claim 1.

22. a) providing a slide, The slide is a first film layer comprising a cross-linked gel, the cross-linked gel comprising a detection agent, a fructosyl oxidase, an anti-interference agent, and a peroxidase; a second film layer comprising a gel; a third film layer comprising a lysis agent, a denaturant, and a protease; From bottom to top, the laminate of film layers includes Step, b) contacting the third film layer of the slide with an unlysed blood sample containing red blood cells, wherein the lysing agent releases glycated hemoglobin from the red blood cells, the denaturing agent contacts and denatures the glycated hemoglobin, the protease releases fructosyl peptides from the denatured glycated hemoglobin, the fructosyl peptides reach the first film layer and contact the fructosyl oxidase to release peroxide, and the peroxidase and the peroxide contact the detecting agent to emit a detectable signal; c) measuring the amount of hemoglobin from the blood sample, the step of measuring the amount of hemoglobin comprising reading a reflectance density of the blood sample from the slide at a first wavelength of light; and d) measuring the amount of glycated hemoglobin from the blood sample, wherein measuring the amount of glycated hemoglobin comprises detecting a reflectance density of the detectable signal from the blood sample at a second wavelength of light, wherein the second wavelength of light is different from the first wavelength of light; 1. A single-slide method for detecting hemoglobin and glycated hemoglobin, comprising:

23. the first wavelength of light is 540 nm and the second wavelength of light is 670 nm; 23. The method of claim 22.

24. the dissolving agent is a detergent selected from the group consisting of octylphenol ethoxylate (TRITON® X-100), polyethylene glycol sorbitan monolaurate (TWEEN® 20), sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ether (POE), and octylphenoxypolyethoxyethanol (NONIDET® P-40); 23. The method of claim 22.

25. The detergent is octylphenol ethoxylate.

25. The method of claim 24.

26. The denaturant is one or more of sodium nitrite or N-lauroyl sarcosine (NLS).

23. The method of claim 22.

27. The protease is a metalloprotease, an endoprotease, or an exoprotease.

23. The method of claim 22.

28. The metalloprotease is a neutral protease.

28. The method of claim 27.

29. contacting the fructosyl peptide with an oxidase cofactor that is flavin adenine dinucleotide (FAD); 23. The method of claim 22.

30. The peroxidase is horseradish peroxidase.

23. The method of claim 22.

31. the disclosing agent is a leuco dye; 23. The method of claim 22.

32. a) providing a slide according to claim 1; b) contacting the third film layer of the slide with a blood sample containing red blood cells, wherein the lysing agent releases glycated hemoglobin from the red blood cells, the denaturing agent contacts and denatures the glycated hemoglobin, the protease releases fructosyl peptides from the denatured glycated hemoglobin, the fructosyl peptides cross the second film layer, the fructosyl peptides reach the first film layer and contact the fructosyl oxidase to release peroxide, and the peroxidase and the peroxide contact the detecting agent to emit a detectable signal; and c) measuring the amount of glycated hemoglobin from the blood sample, wherein measuring the amount of glycated hemoglobin comprises detecting a reflectance concentration of the detectable signal in the blood sample; Including, The reflective material portion includes a metal. A single-slide method for the direct detection of glycated hemoglobin.

33. The fructosyl peptide crosses the cross-linked gel.

33. The method of claim 32.

34. The protease is a metalloprotease, an endoprotease, or an exoprotease.

33. The method of claim 32.

35. The metalloprotease is a neutral protease.

35. The method of claim 34.

36. contacting the fructosyl peptide with an oxidase cofactor that is flavin adenine dinucleotide (FAD); 33. The method of claim 32.

37. The peroxidase is horseradish peroxidase.

33. The method of claim 32.

38. the disclosing agent is a leuco dye; 33. The method of claim 32.

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