Device and system for detecting misfolded proteins in biological sample

Abstract

A system for detecting misfolded proteins or aggregates thereof in a biological sample, the system comprising: (a) a light absorption or light reflection detection unit, and / or a camera unit; (b) a container comprising a liquid biological sample, the biological sample containing or possibly containing misfolded proteins or aggregates thereof, the liquid biological sample being a urine sample or placental tissue sample from a pregnant woman; the container is placed in a detection zone or a camera zone of the system. The systems may be used to diagnose and predict diseases characterized by misfolded proteins, for example, to diagnose whether a pregnant woman has or is at risk of having preeclampsia. The system is implemented with a device (100) for detecting misfolded proteins in a biological sample. In some embodiments, a device (100) includes a housing (102), which may include one or more fluid chambers. The housing (102) defines a fluid receiving chamber (104), a fluid separation chamber (106), and a fluid collection chamber (108).

Description

Devices and systems for detecting misfolded proteins in biological samples Technical Field

[0001] The present application relates to biological detection technology, and more particularly, to a device and method for detecting misfolded proteins in biological samples. Background Art

[0002] Preeclampsia is a disease related to the pregnancy process. Currently, little is known about its pathogenesis. It usually refers to the development of hypertension and proteinuria in pregnant women with normal blood pressure before pregnancy after 20 weeks of pregnancy, accompanied by symptoms such as headache, dizziness, nausea, vomiting, and upper abdominal discomfort.

[0003] Currently, the clinical diagnosis of preeclampsia relies primarily on clinical features such as hypertension and proteinuria. Researchers have been dedicated to developing new biomarkers and methods for the diagnosis of preeclampsia. Researchers at Yale University in the United States have discovered that misfolded protein molecules in maternal urine are significantly associated with the development of preeclampsia. These misfolded proteins selectively bind to Congo red dye, which also binds to cellulose. As a result, they developed a spot diffusion-based assay for detecting misfolded proteins in maternal urine for the diagnosis or prediction of preeclampsia.

[0004] However, the spot diffusion technique used in existing technologies is difficult to effectively distinguish positive from negative samples near the critical value, which can easily lead to misjudgment. Therefore, there is a need for an improved detection method and device. Furthermore, there is also a need in the art for detection methods and devices that can perform quantitative analysis and / or have improved sensitivity, specificity, and accuracy.

[0005] Summary of the Invention

[0006] The present disclosure satisfies at least one of the above needs. One object of the present application is to provide a device and method that can effectively detect misfolded proteins.

[0007] In one aspect of the present application, a method for detecting misfolded proteins or aggregates thereof in a biological sample is provided, comprising: (a) providing a biological sample; (b) mixing the biological sample with a detection reagent capable of binding to the misfolded protein or its aggregate; (c) contacting the biological sample mixed with the detection reagent with and passing through a separation matrix, the separation matrix being constructed to adsorb detection reagents that are not bound to the misfolded protein or its aggregate and allowing detection reagents that are bound to the misfolded protein or its aggregate to pass through; (d) collecting the biological sample that passes through the separation matrix in a free liquid state; and (e) detecting the presence of the detection reagent in the collected biological sample in a free liquid state, wherein the presence of the detection reagent indicates the presence of the misfolded protein or its aggregate in the biological sample. In one embodiment, in step (d), the biological sample is collected in a fluid collection chamber, and the collected biological sample is in a free liquid state in the fluid collection chamber. In a further embodiment, the collected biological sample can flow freely in the fluid collection chamber. In still further embodiments, the volume of liquid capable of free flow in the fluid collection chamber is at least about 50 μL, at least about 100 μL, at least about 500 μL, at least about 50 μL, at least about 1 mL, at least about 3 mL, or about 3 mL to about 5 mL.

[0008] In another aspect of the present application, a device for detecting misfolded proteins or aggregates thereof in a biological sample is provided, the device comprising: a shell defining a fluid separation chamber and a fluid collection chamber; wherein the fluid separation chamber is used to receive the biological sample mixed with a detection reagent, wherein the detection reagent is capable of binding to the misfolded protein or aggregates thereof in the biological sample, and the fluid separation chamber is also used to separate and retain the detection reagent that is not bound to the misfolded protein or aggregates thereof in the biological sample from the biological sample mixed with the detection reagent, and allow the detection reagent that is bound to the misfolded protein or aggregates thereof in the biological sample to flow through; and wherein the fluid collection chamber is used to collect the biological sample flowing through the fluid separation chamber.

[0009] In another aspect of the present application, a kit is provided, which includes the device for detecting misfolded proteins in a biological sample according to the aforementioned aspect.

[0010] In yet another aspect of the present application, there is provided use of the device disclosed herein in preparing a kit for diagnosing or prognosing a disease characterized by misfolded proteins.

[0011] In yet another aspect of the present application, a method of predicting the risk of developing a disease characterized by misfolded proteins is provided.

[0012] In another aspect of the present application, there is provided the use of a combination of (1) a detection reagent capable of specifically binding to a misfolded protein or its aggregates and (2) an adsorption medium capable of adsorbing the detection reagent in a free state in the preparation of a device or kit for diagnosing or predicting a disease characterized by misfolded proteins.

[0013] The above is a summary of the present application, which may contain simplifications, generalizations, and omissions of details. Therefore, those skilled in the art should recognize that this section is illustrative only and is not intended to limit the scope of the present application in any way. This summary is neither intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The above and other features of the present application will be more fully understood through the following description and the appended claims, taken in conjunction with the accompanying drawings. It should be understood that these drawings only depict several embodiments of the present application and, therefore, should not be construed as limiting the scope of the present application. The present application will be more clearly and fully illustrated by the accompanying drawings.

[0015] FIG1 shows a device 100 for detecting misfolded proteins in a biological sample according to one embodiment of the present application;

[0016] FIG2 shows an apparatus 200 for detecting misfolded proteins in a biological sample according to one embodiment of the present application;

[0017] 3a-3b show an apparatus 300 for detecting misfolded proteins in a biological sample according to one embodiment of the present application;

[0018] 4a-4c show an apparatus 400 for detecting misfolded proteins in a biological sample according to one embodiment of the present application;

[0019] FIG5 shows a method 500 for detecting misfolded proteins in a biological sample according to one embodiment of the present application.

[0020] Figure 6 shows the test results of the simulated samples. The left side shows the collected liquid of the simulated positive sample; the right side shows the collected liquid of the simulated negative sample.

[0021] FIG. 7 shows the results of alumina filtration urine color test.

[0022] FIG8 shows the detection sensitivity test of aluminum oxide as a separation material.

[0023] Figures 9a-9c show the results of testing simulated samples using cellulose acetate (Figure 9a), dried egg white (Figure 9b), and microcrystalline cellulose (Figure 9c) as separation media. The left side of each figure shows the collected fluid of a simulated negative sample; the right side shows the collected fluid of a simulated positive sample.

[0024] 10a-10c illustrate the process of performing clinical testing using the detection device of the present invention. DETAILED DESCRIPTION

[0025] As used herein, the terms "substantially", "about" and similar terms are used as terms of approximation rather than terms of degree, and are intended to illustrate the inherent deviations in measured or calculated values ​​recognized by those of ordinary skill in the art. Likewise, it is intended that any numerical range described herein include all subranges of the same numerical precision contained within the range. For example, it is intended that the range of "1.0-10.0" include all subranges between (or including) the minimum value of 1.0 and the maximum value of 10.0, that is, with a minimum value greater than or equal to 1.0 and a maximum value less than or equal to 10.0, for example, 2.4-7.6. It is intended that any maximum numerical limit described herein include all lower numerical limits contained therein, and it is intended that any minimum numerical limit described in this specification include all larger numerical limits contained therein. Therefore, the inventors reserve the right to amend this specification (including the claims) to expressly describe any subrange contained within the range expressly described herein. As used herein, the term "about" refers to a value within 10% above or below the value described.

[0026] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally indicate similar components, unless the context indicates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be employed, and other changes may be made, without departing from the spirit or scope of the subject matter of the present application. It is understood that various configurations, substitutions, combinations, and designs of the various aspects of the present application generally described herein and illustrated in the accompanying drawings may be made, all of which are expressly intended to form a part of the present application.

[0027] Researchers have found that unique non-random cleavage products of SERPINA-1 and albumin are present in urine specimens of women with preeclampsia. The tendency of SERPINA-1 fragments to misfold and form supramolecular aggregates has led people to consider early diagnosis of preeclampsia by detecting misfolded proteins. Further studies have found that misfolded proteins present in the urine of women with preeclampsia can bind to Congo red dye, so the prior art uses a test strip loaded with Congo red dye to detect misfolded proteins indicating preeclampsia. For example, Chinese patent application CN108291905A uses lateral flow chromatography technology to embed Congo red dye in a freeze-dried form on a sample pad. After the urine sample is added, the urine is mixed with the Congo red dye and flows through the capture device. The free Congo red dye is captured by the capture device, and the Congo red dye bound to the target protein (i.e., the misfolded protein) in the urine flows through the capture device into the display strip area. Thus, if the urine contains target protein, red (positive) is displayed in the display strip area. If there is no target protein in the urine, the free Congo red dye is captured by the capture device, so as to display colorless (negative) in the display strip area. Similarly, Chinese patent application CN109342737A has adopted spot diffusion technology on the test paper, that is, after the urine sample is mixed with Congo red dye, a certain amount of urine is applied to the filter paper by capillary. For negative samples, Congo red dye therein is combined with the filter paper to form a concentrated red dot, and for positive samples, because Congo red dye is combined with the target protein, the free Congo red dye is lacked in the sample to form a concentrated red dot on the filter paper, but a uniformly diffused red circle is formed, i.e., spot diffusion. By judging concentrated red dot or diffusion spot, the early diagnosis of preeclampsia can be carried out.

[0028] However, the inventor of the present application has found that the existing test paper for detecting misfolded proteins has defects, and it is not suitable for processing the sample near the critical value.When detecting weak positive sample with test paper, because the amount of the misfolded proteins contained in the weak positive sample is relatively less, therefore may have some Congo red dye can not bind misfolded proteins, and this makes the test paper still have a certain amount of free Congo red dye. Too much free Congo red dye can form concentrated red dots equally, or develops the color in the display bar region, thereby causes erroneous diagnosis result.In addition, for lateral flow chromatography, semi-quantitative results can only be obtained at most, and owing to the limited (μ L level) of sample size that can add, detection specificity and accuracy are also undesirable.

[0029] In order to solve one or more of the above-mentioned problems existing in the prior art, the inventors of the present application abandoned traditional test strips or lateral flow technology based on capillary action or absorption pad suction, and instead creatively performed the detection of misfolded proteins in a liquid state. Specifically, the inventors used a container to load a solution mixed with a biological sample and a dye such as Congo red dye. The color of the collected solution after the mixed solution was treated with a separation matrix can be observed to characterize whether misfolded proteins are present in the biological sample. Generally speaking, the darker the color of the collected solution, the lower the proportion of dye adsorbed by the separation matrix, and the higher the proportion of dye bound to misfolded proteins. The liquid-based detection method and device disclosed in the present invention can not only perform qualitative or semi-quantitative detection through visual inspection, but also allow quantitative detection to be achieved through optical detection equipment. Importantly, the inventors of the present application also unexpectedly found that the liquid-based detection method and device disclosed in the present invention also achieved significantly improved specificity and accuracy.

[0030] Based at least in part on the above findings, the present disclosure provides a liquid-based detection technology.

[0031] In one aspect, a method for detecting misfolded proteins or aggregates thereof in a biological sample is provided, comprising: (a) providing a biological sample; (b) mixing the biological sample with a detection reagent capable of binding to the misfolded protein or its aggregate; (c) contacting the biological sample mixed with the detection reagent with and passing through a separation matrix, the separation matrix being constructed to adsorb detection reagents that are not bound to the misfolded protein or its aggregate and allowing detection reagents that are bound to the misfolded protein or its aggregate to pass through; (d) collecting the biological sample that passes through the separation matrix in a free liquid state; and (e) detecting the presence of the detection reagent in the collected biological sample in a free liquid state, wherein the presence of the detection reagent indicates the presence of the misfolded protein or its aggregate in the biological sample. In one embodiment, in step (d), the biological sample is collected in a fluid collection chamber, and the collected biological sample is in a free liquid state in the fluid collection chamber. In some embodiments, the biological sample can be an untreated or treated biological sample.

[0032] In some embodiments, the biological sample collected in a free liquid state is essentially not attached or adsorbed or bound to the inner wall of the fluid collection chamber, but is able to flow freely in the fluid collection chamber. This is different from the sample flow in lateral flow chromatography, which flows from the sample addition end to the detection end based on capillary action (capillary bed) or the suction of the absorbent pad, wherein the sample in the detection area is bound or restricted by the capillary action or the suction of the absorbent pad. The limited capillary action or the suction of the absorbent pad also limits the sample addition volume allowed by lateral flow chromatography. The use of a capillary bed or an absorbent pad also makes it impossible to perform quantitative detection by optical methods.

[0033] In some embodiments, the collected biological sample is free-flowing in the fluid collection chamber. In some embodiments, the volume of liquid capable of free flow in the fluid collection chamber is at least about 50 μL, at least about 100 μL, at least about 500 μL, at least about 50 μL, at least about 1 mL, at least about 3 mL, or about 3 mL to about 5 mL, for example, about 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, 5.0 mL, 6.0 mL, 7.0 mL, 8.0 mL, 9.0 mL, or 10 mL.

[0034] In some embodiments, the liquid-based detection technology of the present disclosure allows for the addition of a biological sample having a volume of at least about 50 μL, at least about 100 μL, at least about 0.5 mL, at least about 1 mL, at least about 3 mL, or about 3 mL to about 5 mL, for example, about 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, 5.0 mL, 6.0 mL, 7.0 mL, 8.0 mL, 9.0 mL, or 10 mL.

[0035] In some embodiments, the technology based on liquid detection of the present disclosure allows quantitative detection of the collected liquid by optical methods. In some embodiments, at least a portion of the area of ​​the fluid collector is transparent (e.g., a transparent observation window is provided) for the user to observe the color of the mixed solution or to detect by optical methods. The optical method that can be used for the present disclosure is known in the field of optical analysis, including but not limited to spectrophotometry. The optical method that can be used for the present invention can also be selected according to the detection reagent used, for example, when the detection reagent is a fluorescent dye, then fluorescence analysis can be used.

[0036] In one aspect, the present disclosure provides a device for detecting misfolded proteins or aggregates thereof in a biological sample, comprising: a shell defining a fluid separation chamber and a fluid collection chamber; wherein the fluid separation chamber is used to receive a mixture of the biological sample and a detection reagent, wherein the detection reagent is capable of binding to the misfolded proteins or aggregates thereof in the biological sample, the fluid separation chamber contains a separation matrix, which is used to adsorb the detection reagent that is not bound to the misfolded proteins or aggregates thereof from the mixture and allow the detection reagent that is bound to the misfolded proteins or aggregates thereof to flow through; and the fluid collection chamber is used to collect the biological sample that flows through the separation matrix in the fluid separation chamber.

[0037] In some embodiments, detection reagent such as Congo red dye can be pre-loaded in a container before the detection process begins, or can be added to the container (in solid or solution form) during the detection process. In other embodiments, the biological sample can also be mixed with a detection reagent such as Congo red dye in the same container, or can be mixed with a detection reagent such as Congo red dye in advance in other containers.

[0038] In some embodiments, the detection reagents disclosed herein are capable of specifically binding to misfolded proteins or aggregates thereof, and thus can be used as labeling reagents for misfolded proteins or aggregates thereof.

[0039] "Container" or "shell" used in this application refers to an element that can be used to hold a liquid and store or collect a liquid such as a biological sample in a liquid form, and it can have, for example, a structure similar to a graduated cylinder, a beaker, a bottle, a test tube, or any other chamber that can hold a liquid. The configuration of the chamber includes, but is not limited to, a cylindrical, rectangular, conical, hemispherical, or a combination of the above forms. The container or shell may include one or more chambers to be used for different processing operations or steps in the detection process, wherein each chamber can be used for one or more operations or steps. It will be understood that the container or shell can generally be formed of a colorless transparent material, such as glass, acrylic, plastic, polyester, or other materials. Polyester can be, for example, polyvinyl chloride. In some embodiments, a portion of the container can be formed of an opaque material, but at least a portion of the region is transparent (e.g., some transparent observation windows are provided) for the user to observe the color of the mixed solution or to detect it by optical methods.

[0040] The container or shell may include one or more chambers, for example, one or more of a "fluid receiving chamber", a "fluid separation chamber" and a "fluid collection chamber". At least some of the chambers are fluidically connected to each other or can transfer liquids to each other in other forms, for example, by transferring the liquid in one chamber to another chamber by pouring or injecting. Depending on the specific treatment, the chamber may contain a substance or may not contain a substance. In some embodiments, the chamber may contain a detection reagent that can bind to misfolded proteins or other target proteins in a biological sample, such as a dye, or a separation matrix material that can capture or adsorb the dye. In some embodiments, the detection reagent can be, for example, a dye, such as a chemiluminescent dye or a visible light dye or a fluorescent dye, such as an azo dye or its analogs (such as Congo red or Evans blue), a benzothiazole dye or its analogs (such as thioflavin T and thioflavin S), amaranth, brilliant black, Nile red, or a combination of one or more thereof. In some embodiments, the devices and methods of the present application are used for early detection of preeclampsia, and accordingly, the detection reagent that can be used to bind to the misfolded protein of the disease can be an azo dye or its analogue, such as Congo red dye (i.e., 4-amino-3-[4-[4(1-amino-4-sulfonic acid-naphthalene-2-yl)azophenyl]phenyl]azo-naphthalene-1-sulfonic acid disodium). In some embodiments, the detection reagent can be contained in the chamber in solid form, or it can be contained in the chamber in solution form. Preferably, the detection reagent can be stored in solid form, so that when the biological sample is added, the detection reagent is mixed with the biological sample and exists in liquid form, which allows the detection reagent to fully bind to the target protein. The detection reagent stored in non-solution form has long-term stability or a longer shelf life. In one embodiment, the dried detection reagent is Congo red and can be stored in a chamber in an amount of, for example, 0.1 μg-100 μg, more preferably 0.2 μg-50 μg, even more preferably 1 μg to 25 μg (e.g., 2 μg, 5 μg, 10 μg, 15 μg or 20 μg), or stored in a sealed bag and added to the corresponding chamber before or during the detection. The color of the detection reagent is detectable, i.e., visible to the naked eye, and can be detected by visual inspection and / or a mechanical or electronic reader.

[0041] As mentioned above, some chambers may contain a separation matrix material. Due to the specificity of its spatial structure and / or composition, the separation matrix material can compete with the target protein or certain other specific proteins for binding to the detection reagent. It is worth noting that the detection reagent has a lower affinity for the separation matrix material than for the target protein (misfolded protein or aggregates thereof). In other words, if the sample does not contain the target protein, the detection reagent will bind to the separation matrix material, preventing the detection reagent from being retained in the collected biological sample and causing it to develop color; however, if the target protein is present in the biological sample, the detection reagent will bind to the target protein rather than the separation matrix material. As a result, the binding product of the target protein and the detection reagent will be retained in the liquid and flow into the fluid collection chamber, forming an observable colored solution. In some embodiments, the separation matrix material can be any material known to those skilled in the art that can bind to free detection reagent (e.g., containing free hydroxyl groups) through hydrogen bonding or van der Waals forces, or a medium with a microporous surface structure, such as an adsorption medium or a molecular sieve chromatography medium. In some embodiments, the separation matrix does not adsorb or bind to the target protein (i.e., misfolded protein or aggregates thereof). In some embodiments, the separation matrix can be in the form of particles (e.g., having a particle size distribution in the range of about 100-200 mesh, such as about 110 mesh, about 120 mesh, about 130 mesh, about 140 mesh, about 150 mesh, about 170 mesh, about 180 mesh, or about 190 mesh), fibers, or a semisolid (e.g., a gel).

[0042] Separation matrix can be selected based on the affinity of the detection reagent used and the target protein. In some embodiments, the material of the separation matrix may include but is not limited to: cotton (such as cotton or cotton fiber) or cotton gauze; silk or silk cloth; cellulose, such as nitrocellulose, microcrystalline cellulose, cellulose acetate or wood chips; polymers, such as polyester, polyethylene, polysulfone, polyvinyl alcohol, polyethylene glycol (such as PEG2000, PEG3000, PEG4000, PEG5000 or PEG6000) or polyacrylamide; glass fiber; silica gel, gelatin or dextran gel; dry protein or protein powder or a matrix coupled with misfolded protein, such as egg white protein powder; inorganic mineral soil, such as zeolite, clay, kaolin, carboxyapatite, montmorillonite; calcium salts, such as calcium chloride, calcium carbonate or calcium phosphate; activated carbon; or activated alumina; or any combination of the above substances. In some embodiments, the material of the separation matrix is ​​selected from activated alumina, microcrystalline cellulose, cellulose acetate, dry protein or any combination thereof. In some embodiments, the dry protein or dry protein powder may have a particle size of about 0.5-1 mm or larger, and / or may contain congophilic protein or have a beta sheet structure. In one embodiment, the activated alumina is acidic alumina and may have a particle size of about 100-200 mesh, such as about 100-150 mesh.

[0043] In some embodiments, the fluid chambers of the shell can be fluidically connected to each other. Preferably, these fluid chambers can be separated by an isolation layer having micropores or other similar structures, such as a sieve plate, a partition or a woven mesh. The isolation layer can prevent the solid material (such as a separation matrix material) filled in one fluid chamber from being transferred to another fluid chamber, but still allows the liquid to flow between the fluid chambers. In some embodiments, the isolation layer can fix the solid material. In one embodiment, the device of the present disclosure also includes an isolation layer, which is arranged between the fluid separation chamber and the fluid collection chamber, and separates the fluid separation chamber and the fluid collection chamber. The isolation layer allows the biological sample to flow through and prevents the separation matrix from entering the fluid collection chamber. The isolation layer can be in the form of a sieve plate, a partition or a woven mesh, and can be one or more layers. In some embodiments, one or more isolation layers can also be provided on the side of the fluid separation chamber away from the fluid collection chamber to limit the movement of the separation matrix in the fluid collection chamber.

[0044] In some embodiments, the material of the isolation layer does not adsorb biomacromolecules, such as proteins. In some embodiments, the material of the isolation layer having micropores, such as a sieve, partition, or mesh, can include inorganic materials (e.g., inorganic fibers or inorganic particles), such as ceramics, glass, glass fiber, or metals (e.g., stainless steel); polymers, including polymer fibers or particles, such as polyamides (e.g., nylon), polyethylenes (e.g., ultra-high molecular weight polyethylene (UHMW-PE), polytetrafluoroethylene (PTFE), polystyrene, or polyvinyl chloride (PVC)), polyacrylics (e.g., acrylic), polypropylenes, plastics (e.g., porous plastics), polyesters, polyurethanes; cellulose such as filter paper or wood pulp cellulose; or other materials that allow the biological sample to flow through and prevent the separation matrix from entering the fluid collection chamber.

[0045] In some embodiments, the isolation layer is made of porous plastic. Porous plastic, also known as foamed plastic, is a material with a fine-pored, cotton-like structure formed by foaming a synthetic resin matrix with a foaming agent and other additives. Commonly used resins include polyethylene, polystyrene, polyvinyl chloride, polypropylene, and polyurethane.

[0046] In some embodiments, the material of the isolation layer is hydrophilic or hydrophobic. A hydrophobic material can be rendered hydrophilic by surface modification with a hydrophilizing agent or plasma treatment.

[0047] In some embodiments, the isolation layer can be one or more layers. In some embodiments, the isolation layer can have a thickness of about 0.1-5 mm, such as about 0.5-4 mm, about 1-3 mm, about 1.5-2.5 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2.0 mm, about 2.3 mm, or about 3.2 mm.

[0048] Depending on the size of the separation matrix used, the isolation layer material can have a pore size of, for example, about 5 μm to 200 μm, such as 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, or 190 μm. The pore size of the isolation layer material can also be less than 5 μm or greater than 200 μm, as long as the separation matrix is ​​prevented from entering the fluid collection chamber and the sample containing the misfolded protein or its aggregates is allowed to enter the fluid collection chamber.

[0049] In an additional embodiment, the present disclosure provides a porous isolation element that is constructed to allow a biological sample to flow through and prevent the separation matrix from entering. In some embodiments, the porous isolation element is a sieve plate. In some embodiments, the porous isolation element is prepared by a material selected from the following: an inorganic material (e.g., an inorganic fiber or an inorganic particle), such as a ceramic, glass, glass fiber, or a metal (e.g., stainless steel); a polymer, including polymer fibers or particles, such as a polyamide (e.g., nylon), a polyethylene (e.g., ultra-high molecular weight polyethylene (UHMW-PE), polytetrafluoroethylene (PTFE), polystyrene, or polyvinyl chloride (PVC)), a polyacrylic (e.g., acrylic), a polypropylene, a plastic (e.g., a porous plastic), a polyester, a polyurethane; a cellulose such as filter paper or wood pulp cellulose; or any combination thereof. In some embodiments, the porous separator has a thickness of about 0.1-5 mm, such as about 0.5-4 mm, about 1-3 mm, about 1.5-2.5 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2.0 mm, about 2.3 mm, about 3.2 mm. In some embodiments, the porous separator can have a pore size of, for example, about 5 μm to 200 μm, such as 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, or 190 μm.

[0050] In the present application, the target protein capable of binding to the detection reagent in the biological sample can be, for example, a misfolded protein or an aggregate thereof or a supramolecular protein aggregate, or a mixture of the aforementioned substances and a fragment of each protein. The target protein may comprise a β-sheet structure. In addition, the target protein may be a Congo red dye-loving protein. Misfolded proteins may include, for example, α-1 antitrypsin (SerpinAl), ceruloplasmin, heavy chain IgG, light chain IgG, interferon-induced protein 6-16 (IF16-6, G1P3), albumin, a mixture thereof or a fragment thereof or a fragment of each protein. However, misfolded proteins are not limited to the aforementioned proteins. For example, a misfolded protein may be any protein that causes or is associated with a protein misfolding disorder, or a combination of proteins.

[0051] The detection methods and devices of the present application can be used to detect misfolded proteins, aggregated proteins, and / or supramolecularly aggregated proteins in a biological sample, wherein the biological sample is from a mammal, including but not limited to a human, a primate, or a genetically engineered mammal, and the mammal can be pregnant. The detection methods, devices, and kits herein can be used to indicate or predict a subject's risk of developing eclampsia or preeclampsia.

[0052] The biological sample can be a liquid, such as urine, blood, saliva, tissue / interstitial fluid, serum, plasma, cerebrospinal fluid, amniotic fluid. In other embodiments, the biological sample can be a solid and is processed into a liquid before detection. The detection method of the present application can be used for biological samples from mammals. In the case of humans, the method can be used for pregnant women with a gestational age of about 8 to 42 weeks, preferably a gestational age of about 18 to 41 weeks, more preferably about 20 to 41 weeks or 20 weeks to delivery. However, the detection method of the present application can also be used for postpartum mammals. At least one target protein detected by the liquid-based detection device and method disclosed herein can be detected by visual detection to obtain qualitative or semi-quantitative results or by optical methods to obtain qualitative results. For example, the darker the color of the biological sample obtained by separation and collection of the separation matrix, the higher the target protein content in the biological sample, and the greater the possibility that the patient being tested suffers from the related disease.

[0053] The detection device of the present application can also be used to detect protein misfolding disorders other than eclampsia and preeclampsia (including severe preeclampsia and atypical preeclampsia). For example, the device can be used to detect misfolded proteins in the following misfolded protein disorders or conditions, such as Alzheimer's disease, cerebral beta-amyloid angiopathy, retinal ganglion cell degeneration in glaucoma, prion diseases, Parkinson's disease and other synucleinopathy, Tauopathies, frontotemporal lobar degeneration (FTLD), FLTD-FUS, amyotrophic lateral sclerosis (ALS), Huntington's disease and other triads, duplication disorders, dementia (British and Danish familial), hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexander disease, various amyloidoses, Serinopathies, type II diabetes, inclusion body myositis / myopathy, cataracts, retinitis pigmentosa with rhodopsin mutation, medullary thyroid carcinoma, pituitary prolactinoma, hereditary lenticular corneal dystrophy, Mallory bodies (Mallory bodies), body), pulmonary alveolar proteinosis, odontogenic tumor amyloidosis, cystic fibrosis, sickle cell disease, and critical illness myopathy.

[0054] In some embodiments, the detection devices and methods of the present application can provide test results in about 10 minutes or less, about 5 minutes or less, and preferably about 3 minutes or less.

[0055] The present application also includes a test kit for detecting a target protein in a biological sample. The test kit may include a device for detecting misfolded proteins in a biological sample disclosed herein, and / or components required for other detections. For example, the test kit may also include a device for adding the biological sample to the housing, such as a pipette or dropper and instructions for use of the device. In some embodiments, the test kit may also include a color comparison table with different color depths so that the user can judge according to the actual test result (color) by comparing the table. The device can not only be used as an independent entity, but can also be used in the form of a test kit, which is more advantageous in some clinical or non-clinical environments. The test kit can be packaged in a box or a polyester film bag. The test kit bag can also be packaged individually or in multiple packages, for example, 2, 5, 10, 15, 25, 50 or 100 test kits per package.

[0056] In some embodiments, the pipette or dropper is constructed as a fluid receiving chamber, in which a detection reagent is accommodated. The detection reagent can be in powder or granular form. In this way, after the fluid receiving chamber receives the biological sample, the detection reagent stored therein can be mixed and dissolved in the biological sample as expected, thereby being able to fully combine with the target protein in the biological sample. The detection reagent can also be in other solid forms. For example, the fluid receiving chamber can have a matrix material (different from the separation matrix material described herein), such as a sponge or similar structure, and the detection reagent can be dispersedly attached to the matrix material. When the biological sample is added to the fluid receiving chamber, the detection reagent attached to the matrix material can quickly dissolve into the biological sample. Alternatively, the detection reagent can also be in solution form, and after the fluid receiving chamber receives the biological sample, the solution of the detection reagent can also be quickly and fully mixed with the biological sample. In some embodiments, a control line (such as a positive control, such as a β sheet) can also be set in the pipette or dropper.

[0057] In one aspect, the present disclosure provides a liquid aspiration or dripping device, comprising a chamber, wherein the chamber comprises, in sequence in the length direction: (a) an opening, (b) a first isolation layer, (c) a detection reagent layer, (d) optionally, a second isolation layer, and (e) an aspiration element, which is configured to provide a driving force for aspirating or discharging liquid, wherein the detection reagent layer is embedded in the first isolation layer and / or the second isolation layer (if present). In some embodiments, the detection reagent layer is formed by applying a detection reagent solution to the isolation layer and then drying it. In some specific embodiments, drying is air drying at room temperature, while in other preferred embodiments, drying is drying at a temperature range of about 40-70°C, for example, drying at a temperature range of about 50-65°C, or about 55-60°C. The detection reagent layer of the present disclosure is stably attached due to being embedded in the isolation layer, and is quickly released from the isolation layer after contact with the aspirated liquid sample. In some embodiments, the chamber further comprises a quality control layer or a positive control layer, which is disposed between (a) the opening and (e) the aspiration element. In some embodiments, the materials of the first isolation layer and the second isolation layer may be the same or different and may be independently selected from the materials described in the present disclosure that can be used as an isolation layer disposed between the fluid separation chamber and the fluid collection chamber. In some embodiments, the material of the first isolation layer and / or the second isolation layer may also be the same or different from the material of the isolation layer disposed between the fluid separation chamber and the fluid collection chamber. In some embodiments, the first isolation layer and / or the second isolation layer may have a thickness of about 0.1-5 mm, for example, about 0.5-4 mm, about 1-3 mm, about 1.5-2.5 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2.0 mm, about 2.3 mm, about 3.2 mm. The isolation layer of the present disclosure is porous. In some embodiments, the isolation layer material can have a pore size of, for example, about 5 μm-200 μm, for example, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm or 190 μm. In some embodiments, the detection reagent layer of the present disclosure can have a thickness of about 1-5 mm, such as about 1.5-4.5 mm, about 2-4 mm, about 2.5-3.5 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2.0 mm, about 2.3 mm, about 3.2 mm, about 3.7 mm, or about 3.8 mm. In some embodiments, the suction element has a flexible cavity that can accommodate air and provide a driving force by pressing.The liquid aspiration or droplet device of the present disclosure can be used as the sample receiving chamber of the detection device of the present disclosure.

[0058] In one aspect, the present disclosure provides a system for detecting misfolded proteins or aggregates thereof in a biological sample, comprising (a) a light absorption or light reflection detection unit, and / or an imaging unit; and (b) a container, wherein the container contains a liquid biological sample, and the biological sample contains or may contain misfolded proteins or aggregates thereof. In some embodiments, the container is placed in a detection area or an imaging area of ​​the system. In some embodiments, the liquid biological sample is a urine sample or a placental tissue sample from a pregnant woman. In some embodiments, the system of the present disclosure further comprises a separation unit, in particular, the detection device of the present disclosure as a separation unit.

[0059] In another aspect, the present disclosure provides a system for detecting misfolded proteins or aggregates thereof in a biological sample, comprising: (a) a light absorption or light reflection detection unit, and / or an imaging unit; and (b) a detection device of the present disclosure. In some embodiments, the device is placed in a detection area or imaging area of ​​the system.

[0060] The system disclosed herein not only enables qualitative detection of misfolded proteins or their aggregates, but also allows for the determination of the amount of misfolded proteins or their aggregates. Data show that the system disclosed herein further significantly improves the sensitivity, specificity, and accuracy of detection of misfolded proteins or their aggregates.

[0061] The detection methods, devices and systems of the present application can be used by medical personnel, non-professional medical personnel or by the patients themselves (when the patients are human) in clinical laboratories (within a hospital or in an out-of-hospital environment), immediate care environments, physician's office laboratories, emergency rooms (e.g., within a hospital), or as near-patient testing devices or point-of-care devices. In addition, the detection methods and devices of the present application can be combined with other diagnostic assays (e.g., immunoassays, such as those that detect other proteins associated with preeclampsia (i.e., biomarkers such as sFlt-1, PLGF, PP-A, PP13, pentraxin, inhibin-A, and soluble endoglin)) and / or other methods or observations commonly used in diagnosing preeclampsia (e.g., blood pressure readings, clinical tests used in diagnosing preeclampsia, including platelet counts, serum creatinine concentrations, serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase), and other signs or symptoms such as weight gain, dizziness, headache, blurred vision, etc.). It will be understood that the present application does not limit the use environment of the detection methods and devices.

[0062] Next, the device and method of the present application are further described in conjunction with specific implementation plans.

[0063] FIG1 shows a device 100 for detecting misfolded proteins in a biological sample according to one embodiment of the present application.

[0064] As shown in Figure 1, the device 100 includes a housing 102, which can include one or more fluid chambers. In the embodiment shown in Figure 1, the housing 102 defines a fluid receiving chamber 104, a fluid separation chamber 106, and a fluid collection chamber 108. These fluid chambers 104, 106, and 108 can be in fluid communication with each other, thereby allowing liquid to flow within the different fluid chambers in a predetermined sequence.

[0065] Specifically, the fluid receiving chamber 104 is located at the uppermost end of the housing 102 and is operably connected to the outside through an adjacent fluid inlet 110 on the housing 102. In some embodiments, a removable sealing plug 112 is installed on the fluid inlet 110. When the device 100 is temporarily not in use and is stored, installing the sealing plug 112 on the fluid inlet 110 can maintain the airtightness of the interior space of the housing 102 and prevent contamination of the interior space. When the device 100 is to be used to test a biological sample, the sealing plug 112 can be removed from the fluid inlet 112, thereby exposing the interior of the fluid receiving chamber 104. In this way, the biological sample or other liquid can be injected into the fluid receiving chamber 104 through the fluid inlet 110. It will be understood that in some embodiments, the fluid inlet 110 can also be sealed by other structures. For example, the fluid inlet 110 can be sealed by a pierceable or removable sealing membrane, which can be pierced or removed to allow liquid injection before using the device 100 for testing.

[0066] In some embodiments, the fluid receiving chamber 104 contains a detection reagent. Preferably, the detection reagent can be in powder or granular form. Like this, after the fluid receiving chamber 104 receives the biological sample, the detection reagent stored therein can be mixed and dissolved in the biological sample as expected, thereby being able to fully combine with the target protein in the biological sample. The detection reagent can also be in other solid forms. For example, a matrix material, such as a sponge or similar structure, can be provided in the fluid receiving chamber 104, and the detection reagent can be dispersedly attached to the matrix material (different from the separation matrix material described below). When the biological sample is added to the fluid receiving chamber 104, the detection reagent attached to the matrix material can be rapidly dissolved in the biological sample. Alternatively, the detection reagent can also be in solution form, and after the fluid receiving chamber 104 receives the biological sample, the solution of the detection reagent can also be rapidly fully mixed with the biological sample.

[0067] The detection reagent and the biological sample are thoroughly mixed while passing through the fluid receiving chamber 104 to enhance the binding between the detection reagent and the target protein in the biological sample. In some embodiments, the fluid receiving chamber may include a structure that slows down the flow of the liquid, such as an hourglass or a serpentine fluid channel, to increase the time it takes for the biological sample to flow through the fluid receiving chamber 104.

[0068] The fluid receiving chamber 104 can be in fluid communication with the downstream fluid separation chamber 106 via a fluid channel 114, allowing the biological sample mixed with the detection reagent to flow into the fluid separation chamber 106. In some embodiments, the fluid channel 114 can be sealed by a hydrolyzable film (the detection reagent is in solid form), which can retain the solid detection reagent in the fluid receiving chamber 104 and prevent it from unintentionally entering the fluid separation chamber 106 before the detection begins. In other embodiments, the fluid channel 114 can also be sealed by a sealing plug or similar fluid sealing structure (the detection reagent can be in solid or liquid form).

[0069] It will be appreciated that the fluid receiving chamber 104 is primarily used to receive and introduce a biological sample and to fully mix it with the detection reagent. In some embodiments, the housing 102 may not include the fluid receiving chamber 104. In this case, the biological sample can be pre-mixed with the detection reagent outside the housing 102. In this way, the mixed biological sample can be directly injected into the housing 102 via the fluid inlet 110, for example, directly into the fluid separation chamber 106. If the target protein is present in the mixed biological sample, the target protein will fully bind to the detection reagent, significantly reducing the amount of free detection reagent in the biological sample.

[0070] Still referring to FIG1 , the fluid separation chamber 106 located downstream of the fluid receiving chamber 104 contains the separation matrix material described herein. When the biological sample flows through the fluid separation chamber 106, the separation matrix material can adsorb the free detection reagent, preventing the free detection reagent from continuing to flow downstream, but does not affect the flow of other components through it. It will be understood that in some embodiments, in order to increase the residence time of the biological sample in the fluid separation chamber 106, the separation matrix material can be stored relatively tightly in the fluid separation chamber 106 to reduce the flow rate. In other embodiments, the fluid separation chamber 106 can also be constructed with a component for slowing the flow rate, such as an hourglass. In some embodiments, the fluid separation chamber 106 can have a fluid path of a predetermined length (e.g., 1 cm, 2 cm, 5 cm, 10 cm, or longer) to ensure sufficient time to process the biological sample.

[0071] As previously mentioned, the separation matrix material can compete with the target protein or certain other specific proteins for binding to the detection reagent. However, the detection reagent's affinity for the separation matrix material is lower than its affinity for the target protein. In other words, the detection reagent bound to the target protein continues to flow downstream with the biological sample, while the remaining free detection reagent is immobilized in the fluid separation chamber 106 by the separation matrix material and no longer migrates downstream.

[0072] Still referring to Figure 1 , after flowing through the fluid separation chamber 106, the biological sample continues to flow downstream. Specifically, the biological sample flows into the fluid collection chamber 108 via the fluid outlet 116 between the fluid separation chamber 106 and the fluid collection chamber 108. In some embodiments, the fluid outlet 116 can be provided with a removable sealing plug.

[0073] The fluid collection chamber 108 is used to collect the biological sample flowing through the fluid separation chamber 106. When there is a target protein bound to a detection reagent in the biological sample, the biological sample collected in the fluid collection chamber 108 will be colored due to the presence of the detection reagent. At least a portion of the fluid collection chamber 108 is light-permeable so that the colored biological sample collected therein can be visually observed or optically detected. In some cases, the amount of biological sample may be small, so the height of the liquid collected in the fluid collection chamber 108 is limited, which affects the observation effect. Accordingly, the fluid collection chamber can be constructed to have an internal cross-section that tapers downward generally in the vertical direction (e.g., a conical cross-section), thereby increasing the height of the liquid in the chamber for easy observation.

[0074] As can be seen from the embodiment shown in FIG1 , during testing, the housing 102 can be positioned so that the fluid receiving chamber 104, the fluid separation chamber 106, and the fluid collection chamber 108 are arranged from high to low. In this way, the gravity of the biological sample liquid itself can drive it to flow downstream, passing through these chambers and collecting in the fluid collection chamber 108. It will be appreciated that in some other embodiments, the device 100 can also have other fluid driving mechanisms to drive the liquid to flow sequentially in the housing 102. For example, a removable fluid pump, piston, or similar mechanism can be provided at the upper end of the upstreammost fluid receiving chamber 104 of the housing 102. After the biological sample is added to the housing 102, the fluid pump or similar mechanism can be attached to the housing and pressurize the housing 102 to cause the biological sample to flow toward the fluid separation chamber 106 and the fluid collection chamber 108.

[0075] FIG2 shows a device 200 for detecting misfolded proteins in biological samples according to another embodiment of the present application. Compared to the device 100 shown in FIG1 , the device 200 shown in FIG2 does not include a fluid receiving chamber in the housing. Accordingly, a detection reagent can be provided separately and pre-mixed with the biological sample outside the housing. In this way, when the mixed biological sample is injected into the device 200 through the fluid inlet 210 of the housing 202, it can directly enter the fluid separation chamber 206, and then be collected in the fluid collection chamber 208 after being processed by the fluid separation chamber 206.

[0076] Depending on the method of use, the detection device of the present application may also adopt other suitable structures. Figures 3a and 3b show a device 300 for detecting misfolded proteins in a biological sample according to another embodiment of the present application, and Figures 3a and 3b are schematic diagrams of the detection device 300 in different states or stages.

[0077] As shown in Figure 3a, the device 300 includes a housing 302, wherein the housing 302 further includes a fluid separation chamber 306 and a fluid collection chamber 308 in fluid communication with the fluid separation chamber 306. Depending on the different stages of the detection process, the fluid collection chamber 308 also serves as a fluid receiving chamber, which will be described in detail below.

[0078] Specifically, the fluid collection chamber 308 can be pre-loaded with a detection reagent. Referring to Figure 3a, when the test is just beginning, the fluid collection chamber 308 is placed above the fluid separation chamber 306. Similar to the fluid receiving chamber 104 shown in Figure 1, the biological sample is injected into the fluid collection chamber 308 via the fluid inlet 310 (the fluid collection chamber 308 is now used as a fluid receiving chamber) and is fully mixed with the detection reagent loaded in the fluid collection chamber 308. Afterwards, under the action of gravity, the mixed biological sample flows from the fluid collection chamber 308 through the fluid channel into the fluid separation chamber 306, and basically completely carries away the detection reagent in the fluid collection chamber 308. In the fluid separation chamber 306, the separation matrix material therein binds and adsorbs the free detection reagent in the biological sample, thereby retaining it therein.

[0079] Next, as shown in FIG3b , after the device 300 is slightly positioned in the orientation shown in FIG3a for a period of time (e.g., 5 seconds, 10 seconds, 20 seconds, 30 seconds, 2 minutes, or longer), the fluid inlet 310 is sealed with a sealing plug 312 to prevent liquid from flowing out of the housing 302. Next, the device 300 is inverted so that the fluid collection chamber 308 is placed below the fluid separation chamber 306. In this way, under the action of gravity, the biological sample will flow from the fluid separation chamber 306 back into the fluid collection chamber 308 via the fluid channel and collect therein. It will be appreciated that since the detection reagent originally in the fluid collection chamber 308 has been substantially completely dissolved in the biological sample and carried into the fluid separation chamber 306, no detection reagent remains in the fluid collection chamber 308. In some embodiments, the amount of detection reagent contained in the fluid collection chamber 308 does not exceed the amount of detection reagent that the fluid separation chamber 306 can retain (i.e., bind).

[0080] In some embodiments, the detection reagent may not be pre-loaded in the fluid collection chamber 308 , but may be pre-mixed with the biological sample outside the device 300 , or added to the fluid collection chamber 308 separately or simultaneously with the biological sample.

[0081] It can be seen that compared with the device 100 shown in FIG. 1 , the device 300 shown in FIG. 3 a and FIG. 3 b has a simpler structure and lower manufacturing and use costs.

[0082] 4a , 4b and 4c show a device 400 for detecting misfolded proteins in a biological sample according to yet another embodiment of the present application, and are schematic diagrams of the detection device 400 in different states or stages.

[0083] As shown in Figures 4a to 4c, compared to the integrated structure shown in Figures 1 to 3b, the housing 402 of device 400 adopts a split structure, comprising multiple independently constructed and separated cavities. However, each cavity has an opening to allow liquid to flow in and / or out. Next, the device 400 will be further described in conjunction with the sequence of actual testing or use.

[0084] As shown in Figure 4a, housing 402 includes a fluid receiving chamber 404. In some embodiments, fluid receiving chamber 404 is used to receive a biological sample and mix a detection reagent added in advance or during detection with the biological sample so that the target protein in the biological sample can fully bind to the detection reagent.

[0085] Continuing with FIG4b , housing 402 further includes a fluid separation chamber 406 containing a separation matrix material. The user can invert fluid receiving chamber 404 to pour the mixed biological sample from fluid receiving chamber 404 into fluid separation chamber 406. In this manner, the free detection reagent is processed by the separation matrix material, separated from the biological sample, and adsorbed and bound to the separation matrix material.

[0086] As shown in Figure 4c, housing 402 also includes a fluid collection chamber 408, which is used to collect biological samples processed with separation matrix materials. The user can invert the fluid separation chamber 406 to pour the mixed biological sample from the fluid separation chamber 406 into the fluid collection chamber 408. If the original biological sample contains a target protein, it will bind to the detection reagent during the detection process and eventually carry the detection reagent and be collected in the fluid collection chamber 408 and develop color therein. It will be understood that in some embodiments, the fluid collection chamber 408 can share a chamber with the fluid receiving chamber, that is, one chamber acts as a fluid receiving chamber at the beginning of the detection and acts as a fluid receiving chamber in the later stage of the detection. In other embodiments, a fluid receiving chamber may not be provided, but the detection reagent and the biological sample may be pre-mixed outside the device 400.

[0087] The device for detecting misfolded proteins in a biological sample according to the above embodiments of the present application may be provided as a kit.

[0088] Figure 5 shows a method 500 for detecting misfolded proteins in a biological sample according to one embodiment of the present application. As shown in Figure 5, the method 500 includes: in step 502, providing a housing, the housing defining a fluid separation chamber and a fluid collection chamber; in step 504, receiving a biological sample mixed with a detection reagent by the fluid separation chamber, wherein the detection reagent is capable of binding to misfolded proteins in the biological sample; in step 506, separating and retaining the detection reagent that is not bound to the misfolded proteins in the biological sample from the biological sample mixed with the detection reagent by the fluid separation chamber, thereby allowing the detection reagent that is bound to the misfolded proteins in the biological sample to flow through; and in step 508, collecting the biological sample that has flowed through the fluid separation chamber by the fluid collection chamber.

[0089] It can be understood that the devices 100-400 in the above embodiments of the present application can be used to implement a method for detecting misfolded proteins in a biological sample.

[0090] Therefore, in one aspect, the present invention provides a method for detecting misfolded proteins or aggregates thereof in a biological sample, the method comprising: providing a shell, the shell defining a fluid separation chamber and a fluid collection chamber; receiving the biological sample mixed with a detection reagent by the fluid separation chamber, wherein the detection reagent is capable of binding to the misfolded protein or aggregate thereof; separating and retaining the detection reagent that is not bound to the misfolded protein or aggregate thereof from the biological sample mixed with the detection reagent by the fluid separation chamber, thereby allowing the detection reagent bound to the misfolded protein or aggregate thereof to flow through; and collecting the biological sample flowing through the fluid separation chamber by the fluid collection chamber.

[0091] In some embodiments, the housing further defines a fluid receiving chamber, and the fluid separation chamber is located between the fluid receiving chamber and the fluid collection chamber. Furthermore, the method further comprises: before the fluid separation chamber receives the biological sample mixed with the detection reagent, the fluid receiving chamber receives the biological sample mixed with the detection reagent, and allows the biological sample to flow into the fluid separation chamber through the fluid receiving chamber.

[0092] In some embodiments, the housing further defines a fluid receiving chamber, the fluid separation chamber is located between the fluid receiving chamber and the fluid collection chamber, and the fluid receiving chamber contains a detection reagent. Furthermore, the method further comprises: before the fluid separation chamber receives the biological sample mixed with the detection reagent, the fluid receiving chamber receives the biological sample and mixes the biological sample with the detection reagent, so that the biological sample mixed with the detection reagent flows into the fluid separation chamber via the fluid receiving chamber.

[0093] In some embodiments, the method further includes: before the fluid separation chamber receives the biological sample mixed with the detection reagent, the fluid collection chamber receives the biological sample mixed with the detection reagent, and allows the biological sample to flow into the fluid separation chamber through the fluid collection chamber.

[0094] In some embodiments, the fluid collection chamber contains a detection reagent. The method further includes: before the fluid separation chamber receives the biological sample mixed with the detection reagent, the fluid collection chamber receives the biological sample and mixes the biological sample with the detection reagent, so that the biological sample mixed with the detection reagent flows into the fluid separation chamber through the fluid collection chamber.

[0095] In some embodiments, the fluid receiving chamber is in fluid communication with the fluid separation chamber.

[0096] In some embodiments, the collected biological sample is able to flow freely in the fluid collection chamber. In some further embodiments, the volume of liquid that can flow freely in the fluid collection chamber is at least about 50 μL, at least about 100 μL, at least about 500 μL, at least about 1 mL, at least about 3 mL, or about 3 mL to about 5 mL.

[0097] In one aspect, the present disclosure also provides a method for predicting a subject's risk of suffering from a disease characterized by misfolded proteins, comprising detecting the presence of misfolded proteins in a biological sample according to the method of the present disclosure or using the device of the present disclosure, wherein the presence of the misfolded proteins indicates that the subject suffers from or is at risk of suffering from a disease characterized by misfolded proteins.

[0098] In addition, the apparatus and methods of the present disclosure can also be used to diagnose preeclampsia or perform a differential diagnosis in a patient with a gestational hypertension disorder or a patient who may be suspected of having preeclampsia, comprising the following steps: a) obtaining a biological sample (such as urine) from a pregnant patient with hypertension, and b) detecting the presence of misfolded proteins in the biological sample using the method or apparatus of the present disclosure, thereby providing or supporting a diagnosis of preeclampsia in the patient or serving as a differential diagnosis. In some embodiments, the present disclosure also includes a method for treating a pregnant mammal suspected of having preeclampsia, comprising: a) detecting the presence of misfolded proteins in a biological sample from a mammal using the method or apparatus of the present disclosure, thereby determining the presence of misfolded proteins indicating that the pregnant mammal has preeclampsia; and b) causing the pregnant mammal to give birth to treat preeclampsia.

[0099] Implementation Plan

[0100] The present disclosure contemplates including but not limited to the following embodiments, and all combinations of features described in the following embodiments.

[0101] Embodiment 1. A method for detecting misfolded proteins or aggregates thereof in a biological sample, comprising:

[0102] (a) providing biological samples;

[0103] (b) mixing the biological sample with a detection reagent capable of binding to the misfolded protein or aggregates thereof;

[0104] (c) contacting the biological sample mixed with the detection reagent with a separation matrix configured to adsorb the detection reagent not bound to the misfolded protein or its aggregate and allow the detection reagent bound to the misfolded protein or its aggregate to pass therethrough;

[0105] (d) collecting the biological sample that has passed through the separation matrix in a free liquid state; and

[0106] (e) detecting the presence of the detection reagent in the collected biological sample in a free liquid state, wherein the presence of the detection reagent indicates the presence of the misfolded protein or aggregate thereof in the biological sample.

[0107] Embodiment 2. The method according to embodiment 1, wherein the misfolded protein or aggregate thereof is selected from misfolded proteins, protein aggregates, supramolecular protein aggregates, misfolded protein fragments, protein aggregate fragments, supramolecular protein aggregate fragments and any combination thereof.

[0108] Embodiment 3. A method according to embodiment 1 or 2, wherein the misfolded protein or aggregate thereof comprises a β-sheet structure.

[0109] Embodiment 4. The method of any one of embodiments 1-3, wherein in step (b), the biological sample mixed with the detection reagent has a volume of at least about 0.5 mL, at least about 1 mL, at least about 3 mL, or about 3 mL to about 5 mL.

[0110] Embodiment 5. The method according to embodiment 4, wherein in step (d), the biological sample is collected in a fluid collection chamber, and the collected biological sample is in a free liquid state in the fluid collection chamber.

[0111] Embodiment 6. The method according to embodiment 5, wherein the collected biological sample is able to flow freely in the fluid collection chamber.

[0112] Embodiment 7. A method according to embodiment 6, wherein the volume of liquid capable of free flow in the fluid collection chamber is at least about 50 μL, at least about 100 μL, at least about 500 μL, at least about 1 mL, at least about 3 mL, or about 3 mL to about 5 mL.

[0113] Embodiment 8. The method of any one of Embodiments 5-7, wherein the fluid collection chamber does not provide capillary action.

[0114] Embodiment 9. A method according to any one of embodiments 5-8, wherein the collected biological sample in a free liquid state is not attached or adsorbed or bound to the inner wall of the fluid collection chamber or is substantially not attached or adsorbed or bound to the inner wall of the fluid collection chamber.

[0115] Embodiment 10. The method according to any one of embodiments 1-9, wherein the biological sample collected in a free liquid state allows the detection of step (e) to be performed by an optical method.

[0116] Embodiment 11. The method according to any one of embodiments 1-10, wherein the detecting of step (e) is performed by visual or optical methods.

[0117] Embodiment 12. The method according to any one of embodiments 1-11, wherein the detection reagent is capable of specifically binding to the misfolded protein.

[0118] Embodiment 13. A method according to any one of embodiments 1-12, wherein the detection agent is a dye, such as a chemiluminescent dye, a visible light dye or a fluorescent dye, such as an azo dye or an analog thereof (such as Congo red or Evans blue), a benzothiazole dye or an analog thereof (such as thioflavin T and thioflavin S), amaranth red, brilliant black or Nile red.

[0119] Embodiment 14. A method according to any one of embodiments 1-13, wherein the separation matrix comprises one or more materials selected from the following groups: cotton or cotton gauze; silk or silk cloth; cellulose, such as nitrocellulose, microcrystalline cellulose, cellulose acetate, or sawdust; polymers, such as polyester, polyethylene, polysulfone, polyvinyl alcohol, polyethylene glycol (such as PEG2000, PEG3000, PEG4000, PEG5000 or PEG6000), or polyacrylamide; glass fiber; silica gel, gelatin or dextran gel; dry protein or protein powder or a matrix coupled with misfolded proteins, such as egg white protein powder; inorganic mineral soils, such as zeolite, clay, kaolin, carboxyapatite, montmorillonite; calcium salts, such as calcium chloride, calcium carbonate or calcium phosphate; activated carbon; or activated alumina.

[0120] Embodiment 15. The method of any one of Embodiments 1-14, wherein the separation matrix comprises activated alumina and the dye is Congo red or an analog thereof.

[0121] Embodiment 16. The method of any one of embodiments 1-15, wherein the biological sample is from a mammal, such as a human, suspected of having or being at risk of having a protein misfolding disease.

[0122] Embodiment 17. The method of embodiment 16, wherein the protein misfolding disease is selected from the group consisting of preeclampsia, severe preeclampsia, atypical preeclampsia, eclampsia, Alzheimer's disease, cerebral beta-amyloid angiopathy, retinal ganglion cell degeneration in glaucoma, prion diseases, Parkinson's disease and other synucleinopathies, tauopathies, frontotemporal lobar degeneration FTLD, FLTD_FUS, amyotrophic lateral sclerosis, Huntington's disease and other triads, duplication disorders, Dementia (British and Danish familial), hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexander disease, various amyloidoses, serinopathies, type 2 diabetes mellitus, inclusion body myositis / myopathy, cataracts, retinitis pigmentosa with rhodopsin mutation, medullary thyroid carcinoma, pituitary prolactinoma, hereditary lenticular corneal dystrophy, Mallory bodies, pulmonary alveolar proteinosis, odontogenic tumor amyloidosis, cystic fibrosis, sickle cell disease, and critical illness myopathy.

[0123] Embodiment 18. A device for detecting misfolded proteins or aggregates thereof in a biological sample, characterized in that the device comprises:

[0124] a housing defining a fluid separation chamber and a fluid collection chamber;

[0125] The fluid separation chamber is used to receive a mixture of the biological sample and a detection reagent, wherein the detection reagent is capable of binding to the misfolded protein or its aggregates in the biological sample, and the fluid separation chamber contains a separation matrix for adsorbing the detection reagent that is not bound to the misfolded protein or its aggregates from the mixture and allowing the detection reagent that is bound to the misfolded protein or its aggregates to flow through; and

[0126] The fluid collection chamber is used to collect the biological sample flowing through the separation matrix in the fluid separation chamber.

[0127] Embodiment 19. The device according to embodiment 18, wherein the misfolded protein or aggregate thereof is selected from the group consisting of misfolded proteins, protein aggregates, supramolecular protein aggregates, misfolded protein fragments, protein aggregate fragments, supramolecular protein aggregate fragments and any combination thereof.

[0128] Embodiment 20. The device of embodiment 18 or 19, wherein the misfolded protein or aggregate thereof comprises a beta-sheet structure.

[0129] Embodiment 21. The device of any one of embodiments 18-20, wherein the detection reagent is capable of specifically binding to misfolded proteins.

[0130] Embodiment 22. A device according to any one of embodiments 18-21, wherein the detection agent is a dye, such as a chemiluminescent dye, a visible light dye or a fluorescent dye, such as an azo dye or an analog thereof (such as Congo red or Evans blue), a benzothiazole dye or an analog thereof (such as thioflavin T and thioflavin S), amaranth, brilliant black or Nile red.

[0131] Embodiment 23. A device according to any one of embodiments 18-22, wherein the separation matrix comprises one or more materials selected from the following groups: cotton or cotton gauze; silk or silk cloth; cellulose, such as nitrocellulose, microcrystalline cellulose, cellulose acetate, or sawdust; polymers, such as polyester, polyethylene, polysulfone, polyvinyl alcohol, polyethylene glycol (such as PEG2000, PEG3000, PEG4000, PEG5000 or PEG6000), or polyacrylamide; glass fiber; silica gel, gelatin or dextran gel; dry protein or protein powder or a matrix coupled with misfolded proteins, such as egg white protein powder; inorganic mineral soils, such as zeolite, clay, kaolin, carboxyapatite, montmorillonite; calcium salts, such as calcium chloride, calcium carbonate or calcium phosphate; activated carbon; or activated alumina.

[0132] Embodiment 24. The device of any one of embodiments 18-23, wherein the fluid collection chamber is configured to collect the biological sample flowing through the fluid separation chamber in a free liquid state.

[0133] Embodiment 25. The device of embodiment 24, wherein the collected biological sample is able to flow freely in the fluid collection chamber.

[0134] Embodiment 26. A device according to embodiment 25, wherein the volume of liquid capable of freely flowing in the fluid collection chamber is at least about 50 μL, at least about 100 μL, at least about 500 μL, at least about 1 mL, at least about 3 mL, or about 3 mL to about 5 mL.

[0135] Embodiment 27. The device of any one of Embodiments 18-26, wherein the fluid collection chamber does not provide capillary action.

[0136] Embodiment 28. A device according to any one of embodiments 18-27, wherein the collected biological sample is not attached or adsorbed or bound to the inner wall of the fluid collection chamber or is substantially not attached or adsorbed or bound to the inner wall of the fluid collection chamber.

[0137] Embodiment 29. The device of any one of embodiments 18-28, wherein at least a portion of the fluid collection chamber is light-transmissive to enable optical detection of a biological sample collected therein.

[0138] Embodiment 30. The device of any one of Embodiments 18-29, wherein the device further comprises an isolation layer disposed between the fluid separation chamber and the fluid collection chamber and separating the fluid separation chamber and the fluid collection chamber.

[0139] Embodiment 31. The device of embodiment 30, wherein the isolation layer allows the biological sample to flow through and prevents the separation matrix from entering the fluid collection chamber.

[0140] Embodiment 32. A device according to embodiment 31, wherein the material of the isolation layer includes materials selected from inorganic materials (such as inorganic fibers or inorganic particles), such as ceramics, glass, glass fibers, or metals (such as stainless steel); polymers, including polymer fibers or particles, such as polyamides (such as nylon), polyethylenes (such as ultra-high molecular weight polyethylene (UHMW-PE), polytetrafluoroethylene (PTFE), polystyrene, or polyvinyl chloride (PVC)), polyacrylics (such as acrylic), polypropylenes, plastics (such as porous plastics), polyesters, polyurethanes; cellulose such as filter paper or wood pulp cellulose, and optionally, the material is hydrophilic or has been hydrophilically treated.

[0141] Embodiment 33. The device of any one of Embodiments 30-32, wherein the isolation layer has a thickness of 0.1-5 mm and / or a pore size of 5-200 μm.

[0142] Embodiment 34. A device according to any one of embodiments 18-33, wherein the biological sample is from a mammal, such as a human, suspected of having or being at risk of having a protein misfolding disease, such as a sample selected from one or more of the following groups: urine, blood, saliva, tissue fluid, interstitial fluid, serum, plasma, cerebrospinal fluid or amniotic fluid.

[0143] Embodiment 35. The device of any one of Embodiments 18-34, wherein the housing further defines a fluid receiving chamber for receiving the biological sample.

[0144] Embodiment 36. The device of embodiment 35, wherein the detection reagent is contained in the fluid receiving chamber and the biological sample is mixed with the detection reagent.

[0145] Embodiment 37. The device of embodiment 35 or 36, wherein the fluid separation chamber is in fluid communication with the fluid receiving chamber or is separate from the fluid receiving chamber.

[0146] Embodiment 38. The device of any one of Embodiments 35-37, wherein the fluid receiving chamber further comprises a member for slowing down the flow of the fluid, such as an hourglass.

[0147] Embodiment 39. The device of any one of Embodiments 35-38, wherein a removable sealing plug is installed at the fluid inlet of the fluid receiving chamber.

[0148] Embodiment 40. The device of any one of embodiments 18-34, wherein the detection reagent is contained in the fluid collection chamber, and the fluid collection chamber further comprises:

[0149] a sample inlet, through which the fluid collection chamber receives the biological sample and mixes the biological sample with the detection reagent contained therein;

[0150] a fluid channel located between the fluid collection chamber and the fluid separation chamber to allow the biological sample mixed with the detection reagent to flow from the fluid collection chamber into the fluid separation chamber, and to allow the biological sample flowing through the fluid separation chamber to flow back into the fluid collection chamber; and

[0151] a sealing plug for removably sealing the sample inlet;

[0152] Wherein, the amount of the detection reagent contained in the fluid collection chamber does not exceed the amount of the detection reagent that can be retained by the fluid separation chamber.

[0153] Embodiment 41. The device of any one of Embodiments 18-34, wherein the fluid collection chamber further comprises:

[0154] a sample inlet, through which the fluid collection chamber receives the biological sample mixed with the detection reagent;

[0155] a fluid channel located between the fluid collection chamber and the fluid separation chamber to allow the biological sample mixed with the detection reagent to flow from the fluid collection chamber into the fluid separation chamber, and to allow the biological sample flowing through the fluid separation chamber to flow back into the fluid collection chamber; and

[0156] A sealing plug is used to removably seal the sample inlet.

[0157] Embodiment 42. The device of any one of Embodiments 18-41, wherein the fluid collection chamber is constructed to have an internal cross-section that tapers downwardly generally in a vertical direction.

[0158] Embodiment 43. Use of the device according to any one of embodiments 18-42 in the preparation of a kit for diagnosing or prognosing a disease characterized by misfolded proteins.

[0159] Embodiment 44. The use according to embodiment 43, wherein the disease is selected from the group consisting of preeclampsia, severe preeclampsia, atypical preeclampsia, eclampsia, Alzheimer's disease, cerebral beta-amyloid angiopathy, retinal ganglion cell degeneration in glaucoma, prion diseases, Parkinson's disease and other synucleinopathies, tauopathies, frontotemporal lobar degeneration FTLD, FLTD_FUS, amyotrophic lateral sclerosis, Huntington's disease and other triads, duplication disorders, dementia (British and Danish familial), hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexander disease, various amyloidoses, serinopathies, type 2 diabetes, inclusion body myositis / myopathy, cataracts, retinitis pigmentosa with rhodopsin mutation, medullary thyroid carcinoma, pituitary prolactinoma, hereditary lenticular corneal dystrophy, Mallory bodies, pulmonary alveolar proteinosis, odontogenic tumor amyloidosis, cystic fibrosis, sickle cell disease and critical illness myopathy.

[0160] Embodiment 45. The use according to embodiment 44, wherein the disease is preeclampsia.

[0161] Embodiment 46. The use according to embodiment 44, wherein the disease is Alzheimer's disease or Parkinson's disease.

[0162] Embodiment 47. A system for detecting misfolded proteins or aggregates thereof in a biological sample, comprising:

[0163] (a) a light absorption or light reflection detection unit, and / or an imaging unit; and

[0164] (b) a container comprising a liquid biological sample, wherein the biological sample contains or may contain misfolded proteins or aggregates thereof;

[0165] Optionally, the container is placed in a detection zone or an imaging zone of the system.

[0166] Embodiment 48. The system according to embodiment 47, wherein the detection unit is configured to determine the presence or amount of misfolded proteins or aggregates thereof in the liquid biological sample by detecting light absorption or light reflection of the liquid biological sample.

[0167] Embodiment 49. A system according to embodiment 47 or 48, wherein the detection unit uses photometry such as colorimetry or spectrophotometry for detection, and / or the detection unit is a photometer, for example a colorimeter or a spectrophotometer such as an ultraviolet spectrophotometer, a visible spectrophotometer, an infrared spectrophotometer, a fluorescence spectrophotometer or a chemiluminescence photometer.

[0168] Embodiment 50. A system according to any one of embodiments 47-49, wherein the system includes an imaging unit configured to photograph the liquid biological sample and determine the presence or content of misfolded proteins or aggregates thereof in the liquid biological sample through the photographed biological sample image.

[0169] Embodiment 51. A system according to embodiment 50, wherein the imaging unit further comprises an image sensor configured to convert the biological sample image into an electrical signal.

[0170] Embodiment 52. A system according to embodiment 51, wherein the image sensor is a charge coupled device (CCD) image sensor or a complementary metal oxide semiconductor (CMOS) image sensor.

[0171] Embodiment 53. A system according to any one of embodiments 47-52, wherein the container is at least partially transparent to allow light absorption or light reflection detection or image capture, optionally, the container is a cuvette.

[0172] Embodiment 54. The system according to any one of embodiments 47-53, wherein the liquid biological sample has previously been added with a detection reagent capable of binding to the misfolded protein or aggregates thereof.

[0173] Embodiment 55. A system according to embodiment 54, wherein the detection reagent undergoes a spectral shift or color change upon binding to the misfolded protein or aggregate thereof.

[0174] Embodiment 56. A system according to embodiment 54 or 55, wherein the liquid biological sample to which the detection reagent has been previously added has undergone a separation step, wherein the separation step allows the detection reagent to be removed from the liquid biological sample when misfolded proteins or aggregates thereof are not present in the liquid biological sample.

[0175] Embodiment 57. The system of embodiment 56, wherein the separation step comprises passing the liquid biological sample to which detection reagent has been previously added through a chromatography column.

[0176] Embodiment 58. The system of embodiment 57, wherein the chromatography column is a size exclusion chromatography column, and the liquid biological sample corresponding to the retention time of the detection reagent bound to the misfolded protein or aggregate thereof is collected.

[0177] Embodiment 59. A system according to embodiment 56, wherein the separation step includes contacting the liquid biological sample to which the detection reagent has been previously added and passing it through a separation matrix, wherein the separation matrix is ​​constructed to adsorb the detection reagent that is not bound to the misfolded protein or its aggregates and allow the detection reagent that is bound to the misfolded protein or its aggregates to pass through.

[0178] Embodiment 60. A system according to embodiment 59, wherein the separation matrix comprises one or more materials selected from the group consisting of: cotton or cotton gauze; silk or silk cloth; cellulose, such as nitrocellulose, microcrystalline cellulose, cellulose acetate, or sawdust; polymers, such as polyester, polyethylene, polysulfone, polyvinyl alcohol, polyethylene glycol (such as PEG2000, PEG3000, PEG4000, PEG5000 or PEG6000), or polyacrylamide; glass fiber; silica gel, gelatin or dextran gel; dry protein or protein powder or a matrix coupled with misfolded proteins, such as egg white protein powder; inorganic mineral soils, such as zeolite, clay, kaolin, carboxyapatite, montmorillonite; calcium salts, such as calcium chloride, calcium carbonate or calcium phosphate; activated carbon; or activated alumina.

[0179] Embodiment 61. A system according to any one of embodiments 54-60, wherein the detection reagent is a dye, such as a chemiluminescent dye, a visible light dye, or a fluorescent dye, such as an azo dye or its analogues (such as Congo red or Evans blue), a benzothiazole dye or its analogues (such as thioflavin T and thioflavin S), amaranth, brilliant black, or Nile red.

[0180] Embodiment 62. The system of any one of embodiments 47-61, wherein the liquid biological sample is a urine sample or a placental tissue sample from a pregnant woman.

[0181] Embodiment 63. A system according to any one of Embodiments 47-62, wherein the system further comprises a separation unit.

[0182] Embodiment 64. A system according to Embodiment 63, wherein the separation unit comprises a chromatography column, such as a size exclusion chromatography column.

[0183] Embodiment 65. A system according to embodiment 63, wherein the separation unit includes a separation column, which contains a separation medium, which is constructed to adsorb the detection reagent that is not bound to the misfolded protein or its aggregates and allow the detection reagent that is bound to the misfolded protein or its aggregates to pass through.

[0184] Embodiment 66. A system according to embodiment 65, wherein the separation unit comprises the device according to any one of embodiments 8-18, and optionally, the container provides the fluid collection chamber or the fluid collection chamber is used as the container.

[0185] Embodiment 67. A system according to any one of embodiments 20-38, wherein the volume of the liquid biological sample is at least about 50 μL, at least about 100 μL, at least about 500 μL, at least about 1 mL, at least about 3 mL, or about 3 mL to about 5 mL.

[0186] Embodiment 68. A system for detecting misfolded proteins or aggregates thereof in a biological sample, comprising:

[0187] (a) a light absorption or light reflection detection unit, and / or an imaging unit; and

[0188] (b) The apparatus according to any one of embodiments 18-42.

[0189] Optionally, the device is placed in a detection zone or an imaging zone of the system.

[0190] Embodiment 69. The system according to embodiment 68, wherein the detection unit is configured to determine the presence or amount of misfolded proteins or aggregates thereof in the liquid biological sample by detecting light absorption or light reflection of the liquid biological sample.

[0191] Embodiment 70. A system according to embodiment 68 or 69, wherein the detection unit uses photometry such as colorimetry or spectrophotometry for detection, and / or the detection unit is a photometer, for example a colorimeter or a spectrophotometer such as an ultraviolet spectrophotometer, a visible spectrophotometer, an infrared spectrophotometer, a fluorescence spectrophotometer or a chemiluminescence photometer.

[0192] Embodiment 71. A system according to any one of embodiments 68-70, wherein the system includes a camera unit configured to photograph the liquid biological sample and determine the presence or content of misfolded proteins or aggregates thereof in the liquid biological sample through the photographed biological sample image.

[0193] Embodiment 72. A system according to embodiment 71, wherein the imaging unit further comprises an image sensor configured to convert the biological sample image into an electrical signal.

[0194] Embodiment 73. A system according to embodiment 72, wherein the image sensor is a charge coupled device (CCD) image sensor or a complementary metal oxide semiconductor (CMOS) image sensor.

[0195] Embodiment 74. A method for predicting the risk of developing a disease characterized by misfolded proteins, comprising detecting the presence of misfolded proteins or aggregates thereof in a biological sample according to the method of any one of embodiments 1-17 or using the device of any one of embodiments 18-42 or the system of any one of embodiments 47-73.

[0196] Embodiment 75. Use of a combination of (1) a dye capable of specifically binding to a misfolded protein and (2) a separation column containing an adsorbent material capable of adsorbing the dye in a free state in the preparation of a kit for diagnosing or prognosing a disease characterized by misfolded proteins.

[0197] Embodiment 76. The use according to embodiment 75, wherein the separation column is located in a fluid separation chamber defined by the housing, and the housing further defines a fluid collection chamber in fluid communication with the fluid separation chamber.

[0198] Embodiment 77. A liquid aspiration or dripping device, comprising a chamber, which comprises, in sequence in the length direction: (a) an opening, (b) a first isolation layer, (c) a detection reagent layer, (d) optionally, a second isolation layer, and (e) an aspiration element, which is configured to provide a driving force for aspirating or discharging liquid, wherein the detection reagent layer is embedded in the first isolation layer and / or the optional second isolation layer.

[0199] It should be noted that although several steps or modules of the apparatus and method for detecting the presence of misfolded proteins or aggregates thereof in a biological sample are mentioned in the detailed description above, this division is merely exemplary and not mandatory. In fact, according to the embodiment of the present application, the features and functions of two or more modules described above can be concretized in one module. Conversely, the features and functions of one module described above can be further divided into multiple modules to be concretized.

[0200] Example

[0201] Example 1 Dye Test

[0202] Different types of dyes were tested, including thioflavin T, thioflavin S, Congo red, Evans blue, amaranth, brilliant black, and Nile red. The test results are expressed as "excellent," "good," "medium," and "poor" to indicate the applicability of the dye. "Excellent" indicates that the dye can bind to misfolded proteins in the sample, can be separated from free dye, the color is easy to observe, and the test repeatability is good. "Good" indicates that the dye can bind to misfolded proteins in the sample, can be separated from free dye, the color is easy to observe, and the test repeatability is average. "Medium" indicates that the dye can bind to misfolded proteins in the sample, can be separated from free dye, the color is not easy to observe, and the test repeatability is average. "Poor" indicates unusable. The test results are shown in the following table:

[0203] Example 2 Separation Material Test

[0204] Various separation materials were tested. Test results were graded as "excellent," "good," "fair," or "poor" to indicate the suitability of the materials. "Excellent" indicates that the material can distinguish between free and bound dyes, the sample flow rate through the material is appropriate, the material is easy to load, the urine background color can be filtered out, and the test reproducibility is good. "Good" indicates that the material can distinguish between free and bound dyes, the sample flow rate through the material is appropriate, the test reproducibility is good, the material loading is fair, and the urine color cannot be filtered out. "Fair" indicates that the material can distinguish between free and bound dyes, the sample flow rate through the material is appropriate, the test reproducibility is fair, the material loading is fair, and the urine color cannot be filtered out. "Poor" indicates that the material is unusable.

[0205] Example 3 Insulation Material Test

[0206] The following isolation materials were tested. Test results are graded as "Excellent," "Good," "Fair," or "Poor" to indicate the suitability of the materials. "Excellent" indicates that the material provides good isolation and load-bearing performance, a suitable sample flow rate through the material, easy loading, and good test repeatability. "Good" indicates that the material provides good isolation and load-bearing performance, a suitable sample flow rate through the material, good test repeatability, and fair loading ease. "Fair" indicates that the material provides fair isolation and load-bearing performance, a fair sample flow rate through the material, and fair loading ease. "Poor" indicates that the material is unsuitable.

[0207] Example 4 Congo red dye test

[0208] To further test the performance of the dye Congo red, a 1 mg / mL human plasma albumin (HSA) solution was prepared and heated at 90°C for 30 minutes to serve as a simulated positive sample. A 5 mg / mL Congo red solution was prepared as the dye stock solution. A separation column was prepared using 0.2 g of alumina as the separation material. 1 mL of the 1 mg / mL HSA solution was taken as a simulated positive sample, and 1 mL of water was taken as a simulated negative sample. 5 μL of the Congo red dye stock solution was added to each column. The simulated negative and positive samples were passed through the alumina separation column, and the staining of the collected liquid was observed. The results showed that the collected liquid of the simulated positive sample was red, while the collected liquid of the simulated negative sample was colorless, as shown in Figure 6.

[0209] Example 5 Alumina Filtration Urine Color Test

[0210] A separation column was prepared using 0.2g of aluminum oxide as the separation material. 1mL of each negative and positive urine sample was collected and 5μL of a 5mg / mL Congo red solution was added. The samples were passed through the alumina separation column and the color of the collected solution was observed. The results showed that the collected solution for positive samples was red, while the collected solution for negative samples was colorless and transparent, as shown in Figure 7.

[0211] Example 6 Separation Matrix Material Detection Sensitivity Test

[0212] A separation column was constructed using 0.2 g of alumina as the separation material. A gradient solution of denatured human serum albumin (HSA) was prepared (1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL). After heating at 90°C for 30 minutes, 1 mL of each HSA gradient solution was added to 5 μL of a 5 mg / mL Congo red solution. The solution was then passed through the alumina separation column, and the color of the collected solution was observed. The results showed that when the HSA concentration in the sample was 0.25 mg / mL, a distinct red color was visible to the naked eye. At a concentration of 0.125 mg / mL, the collected solution exhibited a light red color, indicating that the detection sensitivity could reach at least 0.25 mg / mL or even lower. The results are shown in Figure 8.

[0213] 0.3g of cellulose acetate, 0.3g of dry protein (duck egg white protein powder), and 0.3g of microcrystalline cellulose were used as separation materials to make separation columns. Human serum albumin HSA solution (10mg / mL) was prepared. After heating at 90℃ for 30 minutes, the denatured HSA was diluted to 0.3mg / mL, a total of 1ml, and 7μL of 5mg / mL Congo red solution was added. Pass through the separation column respectively and observe the color of the collected liquid. The results showed that when denatured HSA was present, obvious red could be seen by naked eye, while when HSA was not present, the effluent was colorless. The results are shown in Figures 9a-9c. The above results also show that the three separation media tested can also achieve a detection sensitivity at least equivalent to that of activated alumina.

[0214] Example 7 Adsorption and separation of Congo red dye by aluminum oxide

[0215] Purpose: The separation of Congo red dye by aluminum oxide, the separation material selected in the present invention, is based on an adsorption mechanism. This experiment was conducted to verify this mechanism.

[0216] Preparation materials: Weigh 4g of anhydrous copper sulfate (Tianmao Chemical, lot: 20200315) and dilute to 250mL with deionized water. The concentration of the resulting copper sulfate solution is 0.1M. Weigh 5mg of Congo red (Sigma, lot: MKBX8167V), add 1mL of deionized water, and shake well to form a uniform solution. The concentration of the prepared Congo red solution is 5mg / mL. To prepare a simulated positive sample, weigh 10mg of human serum albumin (HSA) (Solarbio, lot: 7241052), add 1mL of deionized water, and prepare a 10mg / mL HSA solution. Heat in a boiling water bath at 90℃ for 30min and return to room temperature for use. To prepare a separation device, weigh 0.8g of alumina and add it to a homemade separation column.

[0217] Detection: Copper sulfate solution (a solution without denatured protein) is used to simulate negative samples. The copper sulfate solution itself is blue. After adding Congo red solution, the solution turns nearly black. Take 1 mL of copper sulfate solution, add 7 μL of Congo red solution, shake well, add to the separation column, and after separation by the separation column, the color of the collected liquid is blue. The absorbance of the collected liquid is measured at 490 nm (characteristic absorption band of free Congo red) and 800 nm (characteristic absorption band of copper sulfate solution). The absorbance at 490 nm is 0.001, and the absorbance at 800 nm is 0.021, that is, there is no Congo red in the collected liquid, which means that the Congo red in the above mixed solution is still retained in the alumina separation column, while copper sulfate is in the collected liquid.

[0218] A simulated positive sample (denatured HSA solution) was diluted to 1 mg / mL. 1 mL of the sample was added to 7 μL of Congo red solution and shaken thoroughly. The mixture was red and then added to the separation column. After separation, the collected solution was light red, indicating that some Congo red was not adsorbed by the alumina due to binding to denatured proteins, while the excess free Congo red was adsorbed by the alumina. The collected solution was measured for absorbance at 520 nm, with an absorbance of 0.038, indicating that the collected solution contained bound Congo red dye.

[0219] Conclusion: The aluminum oxide used in the present invention distinguishes between negative and positive samples by separating the free and bound dyes. In this experiment, the blue color of the copper sulfate solution is formed by copper ions, and the relative mass of copper is much smaller than that of the dye Congo red. After the simulated negative sample is separated by the separation column, if the separation mechanism is a molecular sieve effect, Congo red should flow out before the copper ions. However, in this experiment, the collected liquid is blue, and no characteristic absorption formed by Congo red is observed after measurement at 490nm; in the simulated positive sample, the collected liquid is red. This is because the Congo red dye is bound to the denatured protein and is brought into the solution by the denatured protein, causing the solution to appear red, and forming the characteristic light absorption of the bound Congo red dye at 520nm. Based on this, the separation of free dyes by aluminum oxide is based on the adsorption of the dye, rather than the molecular sieve effect.

[0220] Example 8 Clinical Study

[0221] This trial uses a test reagent to test urine samples from subjects. The test results are compared with the gold standard and statistically analyzed to verify the clinical value of the test reagent. The minimum number of subjects in each group should meet the requirements of statistical analysis.

[0222] Clinical trial groups

[0223] 1 Comparison method

[0224] The diagnostic criteria for preeclampsia in the Chinese Medical Association's Guidelines for the Diagnosis and Treatment of Hypertension Complicating Pregnancy (2015) were used as the gold standard.

[0225] 2 Selection of research subjects

[0226] This study is applicable to pregnant women aged 18 years and above, with a gestational age of 20 weeks or more. The subjects were grouped according to the gold standard diagnostic results.

[0227] 2.1 Positive group

[0228] Subjects diagnosed with preeclampsia according to the "Guidelines for the Diagnosis and Treatment of Hypertension Complicating Pregnancy of the Chinese Medical Association (2015)" were enrolled;

[0229] 2.2 Negative group

[0230] Subjects diagnosed with non-preeclampsia diseases according to the "Guidelines for the Diagnosis and Treatment of Hypertension Complicating Pregnancy of the Chinese Medical Association (2015)" were enrolled.

[0231] 3. Selection of clinical trial institutions

[0232] In accordance with the "Regulations on the Registration and Administration of In Vitro Diagnostic Reagents", "Technical Guidelines for Clinical Trials of In Vitro Diagnostic Reagents" and other laws and regulations and the requirements of clinical sample size, while taking into account factors such as epidemiological background and in accordance with the National Drug Clinical Trial Institutions Directory approved by the State Food and Drug Administration, select two or more medical institutions with clinical trial qualifications as clinical research units.

[0233] 4 Selection of clinical samples

[0234] 4.1 Sample type: urine;

[0235] 4.2 Sample collection, storage and transportation

[0236] 1) Select eligible subjects according to the inclusion criteria; 2) Obstetrics department collects urine samples; 3) 5 ml of midstream urine is collected from enrolled subjects and placed in a collection tube; 4) Sample storage and transportation conditions must comply with the instructions; 5) Refrigerated or frozen samples should be slowly returned to room temperature before testing.

[0237] 4.3 Clinical records

[0238] Researchers collect samples from subjects who meet the inclusion criteria and record the subjects' basic information, sample collection date, operator, etc.

[0239] 4.4 Sample selection criteria

[0240] 1) Voluntarily participate in this trial and sign the informed consent; 2) Age ≥ 18 years old; 3) Gestational age ≥ 20 weeks;

[0241] 4.5 Sample Exclusion Criteria

[0242] 1) Patients with obvious macroscopic hematuria; 2) Patients who cannot complete the gold standard diagnosis of preeclampsia.

[0243] 4.6 Sample Rejection Criteria

[0244] 1) Samples with missing traceability information; 2) Samples that are not collected according to the sample collection requirements or are insufficient in quantity should be excluded; 3) Subjects who cannot be tested due to collection, storage, experimental operation errors, etc., and whose samples are difficult to re-collect should be excluded; 4) Samples that are contaminated, corrupted, or degraded by any factors will increase the uncertainty of the test results and should be excluded; 5) Duplicate samples from the same subject should be excluded; 6) Subjects that the researcher believes are not suitable to continue participating in this clinical study.

[0245] 5. Implementation of clinical trials

[0246] 5.1 Sample testing

[0247] Sample preparation

[0248] Collect approximately 3 to 5 mL of midstream urine in a clean container for immediate use or store at 2-8°C until use. Bring the urine sample to room temperature before use.

[0249] Detection kit

[0250] The structure of the detection kit is shown in Figures 10a-9c, in which 0.8 g of acidic alumina was used as a separation matrix, Congo red was used as a detection reagent, and beta sheet was used as a positive control.

[0251] Test steps

[0252] (1) Return the test kit and sample to room temperature before use. (2) Unscrew the cap of the test tube and place the test tube upright on a flat surface (Figure 10a). (3) Fully squeeze the rubber bulb of the pipette, insert the pipette into the urine collected in the container, and draw up the urine to the scale line (Figure 10b). Note: The urine sample will turn red in the pipette. (4) Slowly squeeze the rubber bulb of the pipette to load the urine sample into the separation matrix in the test tube. Re-tighten the cap and let the test tube stand upright for about 3-10 minutes, then observe the color of the eluate in the test tube and the control line in the pipette (Figure 10c).

[0253] Test results

[0254] (1) Negative result: A colorless liquid flows out from the bottom of the test tube. A negative result indicates that the pregnant woman may not have preeclampsia at the time of the test.

[0255] (2) Positive result: The outflow from the lower part of the test tube is red. A positive result indicates that the pregnant woman may have preeclampsia and should consult a doctor for further information.

[0256] (3) Invalid result: In a successful test, the control line in the pipette shown in Figure 10c turns red. If the control line does not turn red, repeat the test with another pipette and test tube. If the outflow is yellow-brown, the urine sample is not suitable for testing and the test result is invalid.

[0257] 5.2 Data Summary

[0258] After the clinical trial is completed, the clinical data will be summarized and submitted to statisticians for statistical analysis.

[0259] 6 Clinical evaluation methods

[0260] The test results of the assessment reagents were compared with the clear interpretation results of the gold standard to observe the sensitivity, specificity, accuracy, negative predictive value and positive predictive value of the reagents.

[0261] Based on a cohort study of 937 subjects, the kit of the present invention achieved a sensitivity of 74.3%, a specificity of 98.4%, an accuracy of 94.7%, a positive predictive value (PPV) of 89.2%, and a negative predictive value (NPV) of 95.5%. Clinical data demonstrate that the kit of the present invention achieves very high specificity and accuracy while maintaining good sensitivity.

[0262] A person skilled in the art can understand and implement other changes to the disclosed embodiments by studying the specification, the disclosed content, the drawings and the appended claims. In the claims, the word "comprising" does not exclude other elements and steps, and the word "a" or "an" does not exclude pluralities. In the actual application of this application, one part may perform the functions of multiple technical features cited in the claims. Any figure marks in the claims should not be understood as limiting the scope. All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated as being incorporated by reference.

Claims

1. A liquid suction or dripping device, comprising a chamber, wherein the chamber comprises, in sequence along the length direction: (a) an opening, (b) a first isolation layer, (c) a detection reagent layer, (d) optionally, a second isolation layer, and (e) an aspiration element configured to provide a driving force for aspirating or expelling liquid, wherein the detection reagent layer is embedded in the first isolation layer and / or the second isolation layer. 2 . The liquid-absorbing or liquid-dropping device according to claim 1 , wherein the detection reagent layer is formed by applying a detection reagent solution to the isolation layer and then drying.

3. The liquid aspiration or dripping device according to claim 1, wherein the chamber further comprises a quality control layer or a positive control layer, which is arranged between (a) the opening and (e) the aspiration element.

4. A system for detecting misfolded proteins or aggregates thereof in a biological sample, comprising: (a) a light absorption or light reflection detection unit, and / or an imaging unit; and (b) a container comprising a liquid biological sample, wherein the biological sample contains or may contain a misfolded protein or an aggregate thereof, and the liquid biological sample is a urine sample or a placental tissue sample from a pregnant woman; Optionally, the container is placed in a detection zone or an imaging zone of the system.

5. The system according to claim 4, wherein the detection unit is configured to determine the presence or amount of misfolded proteins or aggregates thereof in the liquid biological sample by detecting light absorption or light reflectance of the liquid biological sample. Optionally, the detection unit uses photometry such as colorimetry or spectrophotometry for detection, and / or the detection unit is a photometer, for example, a colorimeter or a spectrophotometer such as an ultraviolet spectrophotometer, a visible spectrophotometer, an infrared spectrophotometer, a fluorescence spectrophotometer or a chemiluminescence photometer.

6. The system according to claim 4 or 5, wherein the system comprises an imaging unit configured to photograph the liquid biological sample and determine the presence or content of misfolded proteins or aggregates thereof in the liquid biological sample through the photographed biological sample image.

7. The system according to any one of claims 4 to 6, wherein the liquid biological sample has previously been added with a detection reagent capable of binding to the misfolded protein or aggregates thereof; Optionally, the detection reagent undergoes a spectral shift or color change upon binding to the misfolded protein or aggregate thereof; Optionally, wherein the liquid biological sample to which the detection reagent has been previously added has been subjected to a separation step, wherein the separation step allows the detection reagent to be removed from the liquid biological sample in the absence of misfolded proteins or aggregates thereof, Optionally, wherein the separation step comprises subjecting the liquid biological sample to which a detection reagent has been previously added passing through a chromatography column, optionally wherein the chromatography column is a size exclusion chromatography column, and the liquid biological sample corresponding to the retention time of the detection reagent bound to the misfolded protein or aggregate thereof is collected; Optionally, wherein the separation step comprises contacting the liquid biological sample to which a detection reagent has been previously added with and passing it through a separation matrix, wherein the separation matrix is ​​configured to adsorb the detection reagent that is not bound to the misfolded protein or its aggregates and allow the detection reagent that is bound to the misfolded protein or its aggregates to pass therethrough; Optionally, the separation matrix comprises one or more materials selected from the group consisting of cotton or cotton gauze; silk or silk cloth; cellulose, such as nitrocellulose, microcrystalline cellulose, cellulose acetate, or sawdust; polymers, such as polyester, polyethylene, polysulfone, polyvinyl alcohol, polyethylene glycol (e.g., PEG2000, PEG3000, PEG4000, PEG5000, or PEG6000), or polyacrylamide; glass fiber; silica gel, gelatin, or dextran gel; dry protein or protein powder or a matrix coupled to misfolded proteins, such as egg white protein powder; inorganic mineral soils, such as zeolite, clay, kaolin, carboxyapatite, montmorillonite; calcium salts, such as calcium chloride, calcium carbonate, or calcium phosphate; activated carbon; or activated alumina; Optionally, the detection agent is a dye, such as a chemiluminescent dye, a visible light dye or a fluorescent dye, such as an azo dye or its analogues (such as Congo red or Evans blue), a benzothiazole dye or its analogues (such as thioflavin T and thioflavin S), amaranth, brilliant black or Nile red.

8. The system according to any one of claims 4 to 7, wherein the system further comprises a separation unit; Optionally, wherein the separation unit comprises a chromatography column, such as a size exclusion chromatography column; Optionally, the separation unit comprises a separation column comprising a separation medium configured to adsorb the detection reagent not bound to the misfolded protein or its aggregate and allow the detection reagent bound to the misfolded protein or its aggregate to pass therethrough.

9. The system according to any one of claims 4 to 8, wherein the system further comprises the aspiration or dripping device according to any one of claims 1 to 3, which is configured to aspirate a liquid biological sample.

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