TWI698641B - Device, kit and method for detecting misfolded protein - Google Patents

Abstract

The invention discloses a device, a kit and a method for detecting whether a sample contains misfolded protein or misfolded protein aggregates. The detection device includes a test component and a spotting component. The test component includes a microporous membrane and the spotting component includes a capillary tube. The kit includes dyes that bind to misfolded proteins and microporous membranes. During detection, the sample to be tested is mixed with the dye to form a mixed solution, the mixed solution is sucked by the capillary, and then the capillary outlet is fully contacted with the microporous membrane. The mixed solution is slowly released from the capillary to the microporous membrane and the microporous membrane Pore membrane staining, by observing the color diffusion to determine whether the sample to be tested contains misfolded proteins or misfolded protein aggregates. The detection is carried out by the present invention, and the result can be recognized by naked eyes or instruments, which facilitates the completion of the detection process.

Description

Device, kit and method for detecting misfolded protein

The invention relates to a detection device and a detection method, in particular to a device and a method for detecting misfolded proteins in a biological sample.

The precise and unmistakable natural folding of proteins is a complex and error-prone process, that is, misfolding is prone to occur. Misfolded proteins are proteins formed by misfolding, which are different from the protein formed by natural folding.

Protein misfolding forms a large number of β-sheet structures arranged in reverse. This structure is exposed to the hydrophobic surface, resulting in a high degree of adhesion between the protein molecules, and aggregates with the β-Sheet structure of other misfolded proteins. The protein molecules gathered together serve as the crystallization nucleus and make other unrelated proteins. Co-polymerization to form aggregates of misfolded proteins, which can accommodate almost infinite polypeptide chains (Misfolded Protein Aggregates: Mechanisms, Structures and Potential for Disease Transmission, Semin Cell Dev Biol. 2011 July; 22(5): 482–487.), Form oligomers and fibrous aggregates.

These aggregates have been found to be associated with a variety of neurodegenerative diseases related to the elderly, such as Alzheimer’s disease, Parkinson’s disease, etc. are caused by misfolded proteins, although these proteins are not the same (Alzheimer’s disease amyloid β peptide, α-Synuclein protein of Parkinson’s disease, Huntingtin protein of Huntington’s disease, Prion protein of mad cow disease), which form a β-sheet structure after misfolding, all of which can form The above aggregates have the same configuration.

Recent studies have found that the urine and placental tissues of patients with preeclampsia contain a large number of misfolded proteins and their aggregates, while normal pregnant women do not have such aggregates, thus discovering the onset of preeclampsia and eclampsia and these misfolded proteins and Its aggregates are related. (Buhimischi et al., Science Translational Medicine, 2014, 6(245):245ra92). However, there is currently no rapid POC (Point of Care) detection kit that uses this marker to detect preeclampsia in clinical practice.

In order to solve at least one of the above technical problems, the first aspect of the present invention provides a device for detecting whether a sample contains misfolded protein, including a test component and a spotting component, the test component includes a microporous membrane, and the spotting component includes 1. 2. 3 or more capillaries. Further, the test component includes a first cover plate and a first bottom plate, and the first cover plate includes a microporous membrane.

Further, the device further includes a sample loading component, on which one or more sample slots are provided for holding samples (test samples, negative control samples, and/or positive control samples) and/or dyes. In an embodiment of the present invention, the first cover plate is covered with a microporous membrane.

In an embodiment of the present invention, a test slot is provided on the first cover plate, and the test slot contains a microporous membrane.

According to the present invention, optionally, the spotting part is a hollow box-shaped part containing 1, 2, 3 or more capillaries; optionally, the spotting part is a solid box-shaped part with 1, 2, 3 or more concave holes for placing corresponding 1, 2, 3 or more capillaries; optionally, the spotting part is a spotting plate, including a main body part and a capillary tube, the main part being The plate-shaped member, the capillary is arranged on the side, the number of the capillary is 1, 2, 3 or more. One end of the capillary is a liquid-taking end for sucking and releasing liquid, and the other end is connected with the main body of the spotting plate; optionally, the spotting part is a capillary.

The second aspect of the present invention provides a kit for detecting whether a sample contains a misfolded protein, including the device according to the first aspect of the present invention, and also includes a dye capable of binding the microporous membrane and the misfolded protein. Preferably, the dye is selected From one or more of heterocyclic dyes, Congo Red, Thioflavin, and Evans Blue, more preferably, the dye is Congo Red.

The third aspect of the present invention provides a kit for detecting whether a sample contains misfolded protein, including a microporous membrane and 1, 2, 3 or more capillaries. In an embodiment of the present invention, it also includes a dye capable of binding to the microporous membrane and misfolded protein. Preferably, the dye is selected from one or more of heterocyclic dyes, Congo red, Thioflavin, and Evans blue, more preferably , The dye is Congo red.

Further, the aforementioned kit further includes a control sample, and the control sample includes a negative control sample and/or a positive control sample.

The fourth aspect of the present invention provides a method for detecting whether a sample contains a misfolded protein, including the following steps:

(1) Mix the sample and the dye that can bind to the microporous membrane and misfolded protein to form a mixed solution.

(2) Use capillary tube to suck at least 5μL of mixed solution;

(3) Closely contact the liquid outlet of the capillary with the microporous membrane, so that the mixed liquid in the capillary is sucked out by the microporous membrane and diffuses slowly in the microporous membrane, thereby slowly and completely released into the microporous membrane;

(4) Based on the color of the dye, observe the diffusion of the mixed solution on the microporous membrane to determine whether the sample contains misfolded protein.

Preferably, the mixed liquid enters the capillary through the siphon of the capillary without external force. Preferably 5-25 μL, more preferably 8-15 μL of the mixed solution is sucked into the capillary and completely released into the microporous membrane.

The fifth aspect of the present invention provides a combination for detecting whether a sample contains misfolded protein, including a capillary tube and a microporous membrane, wherein the liquid outlet of the capillary is in contact with and closely adhered to the surface of the microporous membrane.

Further, the capillary contains a certain amount of a mixture of dyes and samples that can bind the microporous membrane and misfolded protein, and the liquid outlet of the capillary is in close contact with and closely attached to the microporous membrane. Preferably, the The dye is selected from one or more of heterocyclic dyes, Congo Red, Thioflavin, and Evans Blue, and more preferably Congo Red. A certain amount means 5 μL or more, preferably 5-25 μL, more preferably 8-15 μL.

Further, the surface of the microporous membrane and the periphery of the liquid outlet of the capillary are stained due to the slow release of a certain amount of Congo red and sample mixture in the capillary from the liquid outlet of the capillary.

Definition of terms

The "misfolded protein" or "misfolded protein" in the present invention is relative to a correctly folded protein. Protein misfolding forms a large number of reversed β-sheet structures. These β-sheet structures are exposed to the hydrophobic surface, resulting in a high degree of adhesion between protein molecules and other misfolding. The β-Sheet structure of the protein naturally gathers, and the gathered protein molecules serve as the crystallization nucleus, allowing other unrelated misfolded proteins to copolymerize to form misfolded protein aggregates (oligomers and fibrous aggregates). In the present invention, the meaning of "misfolded protein" includes these aggregates formed by misfolded protein.

For example, studies have found that the urine and placental tissues of patients with preeclampsia contain a large number of misfolded proteins and their aggregates, which can specifically bind to Congo red. Observation under an electron microscope reveals that these misfolded protein aggregates have Very similar to the fibrous structure of amyloid-like protein, which is not found in normal pregnant women (Buhimschi et al., SCIENCE Translational Medicine, 2014, 6(245):245ra92).

Even if different proteins have no homology in structure and sequence, once the configuration changes and misfolding cannot be repaired or eliminated, they can form a β-sheet structure, β-sheets derived from different proteins The structure can interact to form misfolded protein aggregates containing different proteins, which can specifically bind to Congo red in configuration.

The "microporous membrane" in the present invention refers to a type of film made of microporous materials or covered with microporous materials on the surface. The microporous materials have such functions due to their spatial structure and/or composition. Competing with misfolded proteins for binding dyes, that is, if the sample does not contain misfolded proteins, the dye binds to the microporous material, making it impossible or difficult for the dye to diffuse on the microporous membrane along with the solvent, forming smaller colored spots; If the dye is combined with the misfolded protein, it will not be combined with the microporous material. Therefore, the product of the combination of the dye and the misfolded protein can diffuse on the microporous membrane, thereby forming larger colored spots. The microporous material can be any material known to those skilled in the art to contain a large amount of free hydroxyl groups, such as cellulose, and the microporous membrane is a cellulose membrane, such as filter paper. The dye can be any known to those skilled in the art that has the above characteristics and can specifically bind to the misfolded protein configuration, such as Congo red.

The "capillary tube" in the present invention refers to a hollow tube whose inner diameter of the liquid port does not exceed 3.5 mm or the cross-sectional area of the liquid port does not exceed 9 mm ^2. When the capillary is inserted into the liquid, the liquid can infiltrate the inner surface of the capillary. , It appears that the height of the liquid level in the capillary is higher than the height of the external liquid level, that is, "capillary phenomenon" occurs. When the capillary leaves the liquid, the liquid remaining inside the capillary due to surface tension is not less than 5μL. Hydrophilicity.

The material and texture or treatment of the capillary can be any capillary known to those skilled in the relevant field that can achieve the above-mentioned infiltrating liquid volume.

In the process of detecting the misfolded protein by the speckle diffusion method based on the capillary phenomenon on the above-mentioned microporous membrane, a dye that can specifically bind to the configuration of the misfolded protein, such as Congo red solution, is used to spread the size of the microporous membrane such as filter paper. , To detect whether there is a misfolded protein.

However, the inventor found that if a pipette (such as a pipette, a micropipette, etc.) is used to drop the sample, the droplet directly falls on the filter paper, forming a larger contact surface with the filter paper and quickly spreading on the filter paper. Even negative samples that do not contain misfolded proteins show large color spots on the filter paper, which are often indistinguishable from the color spots of positive samples, resulting in inaccurate detection results, resulting in many false positives and false negatives. Even if the liquid outlet of the pipette or pipette is small, this defect cannot be overcome. The inventor further discovered that if a capillary is used for spotting (rather than dropping samples), a sufficient volume of the mixture of dye and sample is sucked by the capillary, so that the liquid outlet of the capillary is in close contact with the filter paper, so that the mixed liquid in the capillary is caused by the filter paper. The water absorption of the material is slowly released onto the filter paper, which can significantly increase the difference in color spots produced by negative and positive samples, greatly reduce the false positive rate, and significantly improve the detection accuracy.

Detection method and combination

The present invention provides a method for detecting whether a sample contains misfolded proteins or misfolded protein aggregates, including the following steps: (1) Mixing the sample with a dye that can compete with the microporous membrane and the misfolded protein to form a mixed solution, (2) Use a capillary to absorb a certain amount of mixed liquid; (3) Closely contact the liquid outlet of the capillary with the microporous membrane so that the mixed liquid in the capillary is slowly released onto the microporous membrane; (4) Based on the color of the dye, Observe the diffusion of the mixed solution on the microporous membrane to determine that the sample contains misfolded proteins or misfolded protein aggregates. Preferably, when the diffusion area exceeds a reference value, determine that the sample contains misfolded proteins or aggregates of misfolded proteins Things.

On the other hand, the present invention provides a combination or device for detecting whether a sample contains misfolded protein aggregates, which can be used to implement the above detection method. Generally speaking, the assembly includes a capillary tube and a microporous membrane, wherein the liquid outlet of the capillary tube is in contact with and closely adhered to the surface of the microporous membrane.

Specifically, the method and combination of the present invention can be used to detect a variety of samples, such as whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid, pleural and ascites, tears, vaginal secretions, semen, tissue lysates and With the combination, when the sample contains blood and is colored, it can be centrifuged to remove red blood cells or other interference factors before testing. In particular, the method and combination of the present invention are particularly suitable for detecting whether the urine of pregnant women contains misfolded proteins or misfolded protein aggregates, so as to predict, detect, screen or diagnose whether pregnant women have preeclampsia or are at risk of preeclampsia .

Preferably, the microporous membrane in the present invention may be a film made of a material containing free hydroxyl groups and has water absorption. In a preferred embodiment, the microporous membrane is a cellulose membrane made of cellulose containing free hydroxyl groups, such as filter paper, writing paper, printing paper, and label paper.

The dye can be any dye that can specifically bind to misfolded proteins, and this dye should also be able to competitively bind to the material in the microporous membrane (for example, cellulose containing free hydroxyl groups). In a preferred embodiment, the dye is Congo Red. In other embodiments, the dye is Thioflavin or Evans Blue. The dye can be a solid dye that is directly mixed with a sample such as urine and dissolved in a liquid sample, or it can be mixed with a sample such as urine in the form of a solution, and the concentration of the dye such as Congo red in the mixed solution can be 0.01-2 mg/mL , Preferably 0.02 to 1 mg/mL, more preferably 0.05 to 0.5 mg/mL.

In the present invention, the capillary has two ends along the length of the tube, one end is a liquid outlet, and the other end can be open or closed. The inner diameter of the liquid outlet of the capillary is less than or equal to 3.5 mm or the width of the liquid outlet. The area of the cross section does not exceed 9mm ² . The capillary tube should have enough tube length to be able to absorb and hold at least 4 μL of mixed solution, preferably at least 5 μL of mixed solution, at least 8 μL of mixed solution, and more preferably about 8 to 15 μL. In a specific embodiment of the present invention, a certain amount of mixed liquid taken by the capillary is 2-30 μL, 4-30 μL, more preferably, 5-25 μL, 5-20 μL, 4-17 μL, or 5-17 μL, for example It is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 μL; more preferably 8 to 16 μL. Preferably, the capillary tube is substantially the same in thickness along the length of the tube, and the cross section including the liquid outlet can be round or any other regular or irregular shape, such as those shown in FIG. 1. In a preferred embodiment, the inner diameter of the liquid outlet is about 0.5 to 3 mm, 0.7 to 3 mm, preferably 0.9 to 2.8 mm, or the cross-sectional area is about 0.2 to 7 mm ² , preferably about 0.64 to 6.2 mm ² . In a preferred embodiment, the capillary tube sucks the mixed liquid by capillary phenomenon, sucking at least 5 μL of liquid. In a preferred embodiment, after the capillary absorbs the mixed liquid, when the liquid outlet is in contact with the microporous membrane, the liquid is passively released from the capillary into the microporous membrane by the water absorption of the microporous membrane. Preferably, the rate of liquid release into the microporous membrane is not more than 4.5 μL/sec, not more than 4 μL/sec, preferably 0.5-4 μL/sec, such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5 Or 4 μL/sec, more preferably 1 to 3 μL/sec, such as 1.2, 1.6, 1.7, 1.8, 2.3, 2.6 or 2.8 μL/sec.

When the sample does not contain misfolded protein or misfolded protein aggregates, the result obtained by using the method, combination, device or kit of the present invention is negative, that is, the mixed solution formed by the sample and the dye is spotted into the microwell using the capillary tube After being applied to the membrane, no obvious diffusion is formed on the microporous membrane, or in some embodiments, smaller diffusion spots are formed and the diffusion radius is smaller than the reference value. The reference value can be determined by a certain number (for example, 50-100). The maximum value of the radius of the diffuse spot formed by the sample of the patient judged as negative is determined.

When the sample contains misfolded proteins or misfolded protein aggregates, the result obtained by the method, combination, device or kit of the present invention is positive, that is, the mixed solution formed by the sample and the dye is transferred to the microporous membrane by the capillary tube Later, a diffuse spot larger than the negative sample is formed on the microporous membrane. In some embodiments of the present invention, the positive samples produce larger diffusion spots, and the diffusion radius is greater than or equal to a specific reference value, and the reference value can be a diffusion formed by a certain number (for example, 50-100) of positive samples The minimum radius of the spot is determined. It is worth noting that in some sample tests, a "pseudopod"-like diffusion will occur. Even if the radius of the diffusion spot is greater than the diffusion radius of the negative sample, or greater than the above reference value, this type of diffusion is still judged as negative.

In a preferred embodiment, determining whether the sample contains misfolded proteins or misfolded protein aggregates includes comparing the diffusion result of the mixed solution on the microporous membrane with a comparison card, and the comparison card includes at least negative and positive Examples of sample diffusion results; preferably, the comparison card includes at least 1, 2, or 3 examples in FIG. 15A, and at least 1, 2, or 3 examples in FIG. 15B; more preferably, the comparison card includes All 6 examples.

In a preferred embodiment, determining whether the sample contains misfolded protein or misfolded protein aggregates includes passing the diffusion result of the mixed solution on the microporous membrane through an automatic determination system to complete the detection result judgment and output the judgment result. For example, as shown in FIG. 20, the automatic determination system 200 includes a signal acquisition module 204 and a signal processing module 206. Preferably, the automatic determination system 200 may also include a user interaction module 202, wherein the modules are connected through a cable Or connect wirelessly and transmit data or signals. The signal collection module 204 includes an optical signal collector, such as a digital camera or a scanner. When in use, the user sends a signal acquisition command to the signal acquisition module through the user interaction module 202. The signal acquisition module 204 obtains a picture signal of the diffusion situation by taking pictures or scanning the above diffusion results, and then the collected The image signal is transmitted to the signal processing module 206, and the signal processing module compares the collected signal representing the image with the comparison database, and obtains the judgment result based on a specific algorithm and transmits it to the user interaction module 202. Preferably, the comparison database contains a large amount of clinical sample detection data, and the signal processing module performs comparisons through smart algorithms. Optionally, the signal processing module 206 transmits the determination result to a third-party system, such as a hospital's HIS (Hospital Information System) or LIMS (Laboratory Information Management System) system.

Therefore, the present invention provides an automatic detection system for detecting whether there is a misfolded protein in a sample (such as a pregnant woman's urine) or whether the pregnant woman has preeclampsia or is at risk of preeclampsia, including (1) the combination and device of the present invention Or kits, and the above-mentioned automatic determination system. In another specific embodiment, the automatic detection system of the present invention includes (1) the combination, device or kit of the present invention, (2) the signal acquisition module includes an optical signal collector, such as a digital camera or scanner, and (3) The information memory is used to store processed or unprocessed signals collected by the signal acquisition module. The information memory can be a HIS system or a LIMS system. The optical signal collector of the signal acquisition module, such as a digital camera or scanner, transfers the collected signal, that is, the result of the diffusion of the mixed liquid on the microporous membrane (such as in the form of photographs or digital information) to a HIS system Or LIMS system or computer system.

In one embodiment of the combination of the present invention described above, the liquid outlet in the capillary and above is filled with the above mixed liquid, that is, a sample such as urine and a dye that can compete with the microporous membrane and the misfolded protein are mixed to form a mixture. Mixed liquid; preferably, the volume of the mixed liquid is 1-30 μL, more preferably, 1-25 μL, for example about 1 to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 Or 16 μL; more preferably 8-15 μL.

Further, in the above embodiment, since the capillary is in close contact and close contact with the microporous membrane, a certain amount of mixed liquid contained in the capillary is slowly released onto the microporous membrane, thereby forming the micropores around the liquid outlet of the capillary. The above-mentioned diffusion spots are formed on the surface of the film. Examples of diffuse spots include those shown in Figure 15; in a specific embodiment, after the mixed solution is released from the capillary, the dye accumulates at the liquid outlet, does not diffuse and forms dark spots; in another specific embodiment, the mixed After the liquid is released from the capillary, the dye forms a dark spot near the liquid outlet and spreads like a "pseudopod"; in another specific embodiment, the dye forms near the liquid outlet after the mixed liquid is released from the capillary. Light color spreads but contains pseudopods. In another specific embodiment, after the mixed liquid is released from the capillary, smaller red spots are formed at the liquid outlet, but obvious circular diffusion spots are formed around, or irregular diffusion that diffuses outward is formed at the spot. Spots, but no "pseudopods", or form uniform, large circular diffuse spots.

In a preferred embodiment of the detection method of the present invention, the method for detecting whether the sample contains misfolded protein or misfolded protein aggregates includes the following steps: mixing the sample with a dye that can compete with the microporous membrane and the misfolded protein for binding Form the first mixed liquid; suck a certain amount of the first mixed liquid with the first capillary tube, and bring the liquid outlet of the first capillary into close contact with the first microporous membrane so that the first mixed liquid in the first capillary is slowly released to On the first microporous membrane; use the second capillary to suck a certain amount of the negative control solution, that is, the second mixed solution, and closely contact the liquid outlet of the second capillary with the second microporous membrane so that the second capillary in the second capillary The mixed solution is slowly released onto the second microporous membrane; a certain amount of positive control solution, namely the third mixed solution, is sucked by the third capillary, and the liquid outlet of the third capillary is in close contact with the third microporous membrane to make the third The third mixed liquid in the capillary is slowly released onto the third microporous membrane.

Further, based on the color of the dye, observe the diffusion of the first, second, and third mixtures on the microporous membrane to determine whether the sample contains misfolded proteins or misfolded protein aggregates.

The second mixed solution may be a dye solution determined to be free of misfolded proteins, or a biological sample determined to be free of misfolded proteins, such as a mixed solution of negative urine and dye. The third mixed solution may be a dye solution determined to contain misfolded proteins, which may be formed by mixing a positive sample determined to contain misfolded proteins with a dye, or by mixing a misfolded protein positive reference product with a dye. In a preferred solution, the above method can detect multiple (2, 3, 4, 5, 10 or more) samples at the same time, that is, these multiple samples are mixed with the dye to form a mixed solution, and then the capillary spot is used to form a mixed solution. Diffusion spots, and determine whether these samples contain misfolded proteins based on the diffusion spots.

Similarly, the combination of the present invention may also include 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or more capillaries, and the liquid outlet of each capillary is connected to the micropore respectively. The surface of the membrane is in contact and tightly closed, and these capillaries can be fixed together by a connecting bridge to form a spotter composed of a row of capillaries. As shown in FIG. 16, in FIG. 16A, the assembly 160A includes capillary tubes 162, 182, and 192, each with a liquid outlet 164, 184, and 194, and another open nozzle 166, 186, 196, open tube The ports 166, 186, and 196 penetrate the connecting bridge, so that both ends of the capillary are connected to the outside. The connecting bridge 180 serves to support and fix the capillaries 162, 182, and 192, and the liquid outlets 164, 184, and 194 of the combined body 160A are in contact with and tightly attached to the surfaces of the microporous membranes 168, 188, and 198, respectively. As shown in Figure 16A, the capillaries 162, 182 and 192 are each filled with a mixed solution. Preferably, the liquid in the capillary 162 is a mixture of a urine sample and dye of a pregnant woman to be tested, and the liquid in the capillary 182 is a negative control sample or buffer. The liquid in the capillary 192 is a mixed liquid of the positive control sample and the dye.

Fig. 16B shows that the liquid part of the capillary of the assembly of Fig. 16A is absorbed by the microporous membrane; Fig. 16C shows that the liquid in the capillary of the assembly of Fig. 16A is all absorbed by the microporous membrane, and different forms are formed in the microporous membrane. Spot spread results.

Detection device

Another aspect of the present invention provides a device for detecting whether a sample contains misfolded proteins or misfolded protein aggregates.

Combining and quoting the above description and Figures 12, 13, and 14, a specific embodiment of the detection device invention can include a panel with two rows of grooves and a spotting component embedded in the panel and detachable from the panel. Among the two rows of grooves on the panel, each row has one to more (3, 10, 12 or more) grooves, one of which is a sample groove and the other is a test groove. The diameter of the sample tank is 0.3 to 1 cm, most preferably 0.5 cm, and the depth is 0.3 to 1 cm, most preferably 0.5 cm; the sample tank can contain 30-500 μL or more liquid, preferably, there are more than 3 on the panel The sample slot includes a sample slot to be tested, a negative control sample slot, and a positive control sample slot. The sample tank to be tested can be filled with the above-mentioned dye or dye solution or a mixture of the dye and the sample to be tested; the negative control sample tank can be filled with negative control samples; the positive control sample tank can be filled with positive control samples. The sample tank can be sealed with a sealing material. The diameter of the test slot is 0.5-2 cm, most preferably 1.8 cm, and the depth is 0.2-1 cm, most preferably 0.3 cm. The bottom of the test tank is covered with the aforementioned microporous membrane such as filter paper. Generally speaking, the number of test slots on the panel is the same as or more than the sample slots. The spotting parts that are embedded in the panel and can be detached from the panel are described below. Specifically, they contain one or more capillaries, which can be fixed on a beam or column, for example, two or more of the same length The capillaries are fixed on a beam in parallel and side by side, and the spacing between the capillaries corresponds to the spacing between the sample grooves and the test grooves so that multiple capillaries on the spotting part can be inserted into the corresponding multiple sample grooves or corresponding Multiple test slots.

For example, the detection device of the present invention shown in FIG. 2 includes a test component and a spotting component, the test component includes a microporous membrane, and the spotting component includes a capillary tube. Preferably, the number of capillaries is more than two, such as two, three, 4, 5, 6, 7, 8, 9, 10 or more.

In some embodiments of the invention, the test component may be a separate microporous membrane.

In other embodiments of the present invention, the test component is composed of a first cover plate and a first bottom plate, and the first cover plate and the first bottom plate are either detachably or non-detachably connected. In some embodiments of the present invention, the first cover plate and the first bottom plate are non-detachably connected to form a whole. Although the first cover plate and the first bottom plate are no longer physically distinguished, this is still the case for the convenience of description. distinguish.

In some specific embodiments of the present invention, the first cover plate is covered with a microporous film, as shown in FIGS. 4A and B. Optionally, there are 1 or more microporous membranes, for example 2, 3, 4, 5, 10, 20, 50, 100, 200, and the microporous membrane can be of any shape, For example, round, oval, square or irregular shape. Optionally, one microporous membrane can only be used for the diffusion of one sample. More preferably, the microporous membrane is circular and its area is not less than the maximum area that the sample can diffuse. Optionally, 1 microporous membrane can be used for diffusion of multiple samples, such as 2, 3, 4, 5, 10, 20, 50, 100, 200 or more, more preferably The microporous membrane can be divided into a plurality of sections, and each section can be used for diffusion of a sample. More preferably, the section is square, and the area of the inscribed circle is not less than the maximum area that the sample can diffuse.

In a specific embodiment of the present invention, the first cover plate is provided with a test groove, the test groove is a cylindrical groove with a depth of 1/3 to 2/3 of the thickness of the sample loading part, and the groove is provided with micro holes The membrane, the microporous membrane, is circular, with a diameter slightly smaller than the cross-sectional diameter of the groove, and is laid flat on the bottom of the groove, as shown in Figure 4C and D. In a specific embodiment of the present invention, the number of test slots is multiple, such as 2, 3, 5, 10, 15, 20, 30, 50, 100, 200, preferably Ground, each test slot provides a sample for diffusion.

In other embodiments of the present invention, the first cover plate and the first bottom plate are detachably connected, optionally, by providing a snap connection at the edges and/or four corners, and optionally, bonding by an adhesive. The joint connection, optionally, is connected by welding. In a specific embodiment of the present invention, the first cover plate is provided with a round hole, and a microporous film is arranged between the first cover plate and the first bottom plate. When the first cover plate and the first bottom plate are buckled or locked or glued After closing, the circular hole on the first cover becomes a groove, and the bottom of the groove is a microporous film, thereby forming a test groove, as shown in Figs. 4E and F.

In an embodiment of the present invention, the detection device further includes a sample loading component, which is composed of a second cover plate and a second bottom plate, wherein a sample groove is provided on the second cover plate, and the sample groove is a cylindrical groove.

In a preferred embodiment of the present invention, the lower surface of the second cover plate is detachably or non-detachably connected to the upper surface of the second bottom plate; preferably, it is detachably connected, and more preferably by buckling the edges and/or four corners. Connect, either by adhesive bonding or by welding.

In an embodiment of the present invention, the side surface of the first cover plate is non-detachably connected with the side surface of the second cover plate to form a whole, and there is no physical difference, and the side surface of the first bottom plate is not detachably connected to the second bottom plate 22. The sides are connected as a whole, and there is no physical difference.

In an embodiment of the present invention, a diversion groove is provided between the first cover plate and the second cover plate, and the diversion groove is a long groove perpendicular to the center of the first cover plate and the second cover plate. The shape of the cross section is inverted triangle or arc shape, and the purpose of setting the diversion groove is to prevent the liquid in the sample groove from flowing out and entering the test groove to produce deviations in the detection results. With the diversion groove, if the sample overflows or splashes from the sample groove, it can enter the diversion groove along the surface of the second cover and be stored in the diversion groove or flow out along the diversion groove instead of Enter the first cover. Further, when the number of sample slots is more than one, a guide slot can also be provided between two adjacent sample slots to prevent cross contamination between samples.

In another embodiment of the present invention, the side surface of the first cover plate is detachably connected to the side surface of the second cover plate, or the side surface of the second bottom plate is detachably connected to the side surface of the second bottom plate. In another embodiment of the present invention, the side surface of the first cover plate is detachably connected to the side surface of the second cover plate, and the side surface of the first bottom plate is detachably connected to the side surface of the second bottom plate.

In a more preferred embodiment of the present invention, the connection refers to connection by movable parts, for example, connection by soft materials, or connection by hinges, whereby the sample loading part can be folded with the test part.

In one embodiment of the present invention, the shape of the sample-carrying part or the test part can be any shape, preferably one selected from the group consisting of triangles, quadrangles, polygons, ellipses, and circles. More preferably, the sample-carrying part or the test part The shape of the component is axisymmetric.

In a specific embodiment of the present invention, the number of sample slots is the same as the number of test slots;

In an embodiment of the present invention, the sample grooves and the test grooves are arranged in a single-row linear arrangement, a multi-row linear arrangement, or a polygonal arrangement, a circular arrangement, and an arbitrary arrangement. Preferably, they are arranged in a straight line.

In another specific embodiment of the present invention, the sample tank and the test tank are arranged in the same manner.

In some embodiments of the present invention, the sample groove and/or the test groove may have any shape, preferably one selected from the group consisting of triangle, quadrilateral, polygon, ellipse, and circle.

In a specific embodiment of the present invention, the sample groove and the test groove are circular.

In some embodiments of the present invention, the sample tank has a diameter of 0.3 to 1 cm, most preferably 0.5 cm, and a depth of 0.3 to 1 cm, most preferably 0.5 cm.

In some embodiments of the present invention, the diameter of the test slot is 0.5-2 cm, most preferably 1.8 cm, and the depth is 0.2-1 cm, most preferably 0.3 cm.

In a specific embodiment of the present invention, the sample tank and the test tank have the same size.

In one embodiment of the present invention, the spotting part is a hollow box-shaped part containing a capillary tube.

In some specific embodiments of the present invention, the hollow box-shaped component is detachably or non-detachably connected to the sample carrying component and/or the test component. The hollow box component has an opening to facilitate access to the capillary tube. Preferably, the opening is connected to the sample The opening of the slot and the test slot are facing the same, and more preferably, the opening is provided with a cover.

In some embodiments of the present invention, the spotting component is a solid box-shaped component with recessed holes for placing capillaries.

In some specific embodiments of the present invention, the solid box-shaped component is detachably or non-detachably connected to the sample carrying component and/or the test component. Preferably, the solid box-shaped part is integrated with the sample carrying part and/or the test part. More preferably, a recessed hole is directly provided on the sample loading part and/or the test part for placing the capillary tube. Preferably, there is one recessed hole that can accommodate all the capillary tubes; preferably, the recessed hole can be used for placing multiple capillary tubes, more preferably Ground, the number of concave holes is the same as the number of capillaries, the diameter and depth of the concave holes are slightly larger than the capillary, and each concave hole is placed with a capillary. The recessed hole does not cross the sample slot or test slot.

In one embodiment of the present invention, the spotting component is a spotting device, and a capillary is provided on its side. The main body of the spotter is a rectangular parallelepiped. The side of the rectangular parallelepiped is provided with recessed holes. The recessed holes penetrate the rectangular parallelepiped, so that both ends of the capillary tube are in communication with the outside. The capillary is inserted into the recessed holes. A whole. Correspondingly, the sample loading component and/or the test component are provided with concave holes, and the capillary protruding part of the spotting component can be inserted into the corresponding concave holes.

In a specific embodiment of the invention, the number of capillaries is the same as the number of sample slots or test slots, and the arrangement is the same as that of sample slots or test slots. In a specific embodiment of the present invention, there are two or more capillaries, such as three, which are arranged on the same side of the rectangular parallelepiped main body, so that the spotting component presents the shape of a row of capillaries.

In a specific embodiment of the present invention, the side of the spotter where the capillary is provided further includes a positioning pin, a positioning hole is provided on the first cover plate, and a positioning hole is provided on the second cover plate. When the positioning pin is inserted into the second cover plate When positioning the hole, the capillary is inserted into the sample groove to absorb the liquid. When the positioning needle is inserted into the positioning hole of the first cover, the capillary contacts the microporous membrane in the test groove, and the liquid in the capillary is absorbed by the microporous membrane and in the microporous Diffusion occurs on the membrane.

In an embodiment of the present invention, the shape of the positioning pin is selected from one of bullet head shape, cylinder, rectangular column, triangular column, irregular column, arbitrary column or inclined arbitrary column. In a specific embodiment of the present invention, the shape of the positioning pin is a bullet shape.

In one embodiment of the present invention, the positioning needle is a hollow structure.

In one embodiment of the present invention, the position of the positioning needle is on both sides of the spotter or the middle of the capillary.

In one embodiment of the present invention, the number of positioning pins is at least two, preferably two, three, four or more.

In a specific embodiment of the present invention, the number of positioning needles is two, which are respectively arranged on both sides of the spotter.

In a specific embodiment of the present invention, the different outer surfaces of the sample-carrying part and/or the test part are also provided with concave holes, and the arrangement of the concave holes is the same as that of the capillary and positioning pins on the spotter, so that the spotting The device is fixed on the sample loading part or the test part.

In a specific embodiment of the present invention, the spotting device is provided with protrusions or grooves for the purpose of increasing friction and facilitating the handling, insertion or extraction of the spotting device.

In some embodiments of the present invention, one end of the capillary is connected to a device that can generate negative pressure inside the capillary. In one embodiment of the present invention, the device that can generate negative pressure inside the capillary is a rubber ball, similar to a dropper. When in use, pinch the rubber ball while putting the other end of the capillary into the liquid sample and loosen it. The rubber ball, the rubber ball recovers its deformation, so that negative pressure is generated in the capillary, so that the liquid sample enters the capillary faster. In another preferred embodiment of the present invention, the device capable of generating negative pressure inside the capillary is a piston device, such as a syringe, a micropipette, and the like. When in use, first put the other end of the capillary into the liquid, pull the piston manually or rely on an elastic device to pull the piston, so that negative pressure is generated inside the capillary, so that the liquid sample enters the capillary faster. In the embodiment of the present invention, the above-mentioned device for generating negative pressure inside the capillary can also generate positive pressure inside the capillary, thereby pushing the liquid in the capillary to slowly flow out and transfer to the microporous membrane to complete the detection.

In some embodiments of the present invention, the sample loading part, the test part and the spotting part are made of PVC material, PUC material, nylon material, rubber material, acrylonitrile-butadiene-styrene copolymer material (ABS material), glass Material or metal material. Preferably, the metal material is an alloy material, and more preferably, the alloy material is stainless steel.

The detection device of the present invention has at least two core components, including a microporous membrane detection component and a capillary spotting component. Therefore, those skilled in the art can understand the preparation method according to the content disclosed in this specification: For the detection component, the microporous membrane can be fixed on the supporting surface, through the three methods shown in Figure 4, or other options. method. For spotting parts, if there are more than one capillary, for example three, a connecting bridge can be used to connect the capillary. As shown in Figure 1C, the capillary includes a liquid outlet and another open port, passing through the connecting bridge to make another open port It is also connected to the air. The arrangement of the capillaries can be any other way.

Reagent test kit

Another aspect of the present invention provides a kit for detecting whether a sample contains misfolded protein, including a microporous membrane and a capillary.

In some embodiments of the present invention, the kit includes any of the above detection devices.

In a specific embodiment, the detection device in the kit includes a panel with at least two rows of grooves and a spotting component embedded in the panel and detachable from the panel, wherein the two rows of grooves on the panel In each row, there are 1 to more (3, 10, 12 or more) grooves, one of which is a sample slot and the other is a test slot. Preferably, there are more than 3 sample slots on the panel including a sample slot to be tested (containing 30-500 μL or larger volume of the above-mentioned dye or dye solution or a mixture of the dye and the sample to be tested) and a negative control sample slot (Containing at least 30 μL of negative control sample), a positive control sample slot (containing at least 30 μL of positive control sample). The sample tank is sealed with a sealing material. The bottom of the test tank is covered with the aforementioned microporous membrane such as filter paper. The spotting component embedded in the panel and detachable from the panel contains one or more capillaries, two or more capillaries of the same length are fixed in parallel and side by side on a beam, and the distance between the capillaries and the sample slot and The spacing between the test slots is corresponding so that multiple capillaries on the spotting component can be inserted into the corresponding multiple sample slots at the same time to absorb the liquid therein, and then inserted into the corresponding multiple test slots at the same time.

In a specific embodiment of the present invention, the kit includes a dye capable of binding the microporous membrane and misfolded protein. The dye is selected from one or more of heterocyclic dyes, Congo Red, Thioflavin, and Evans Blue, and is preferably Congo Red. In a preferred embodiment of the present invention, the dye is pre-loaded into the sample tank in the form of a solid dry powder or a solution.

In a specific embodiment of the present invention, the kit further includes a control sample, and the control sample includes a positive control sample and/or a negative control sample. In a preferred embodiment of the present invention, the control sample is pre-loaded into the control sample tank.

In a specific embodiment of the present invention, the sample tank is sealed with a sealing material, and the sealing material is selected from tin foil, plastic film, and aluminum foil. The sealing can be done by glue or plastic sealing or any other method capable of sealing.

In a specific embodiment of the present invention, the negative control sample does not contain misfolded protein, and the positive control sample contains misfolded protein. In some specific embodiments of the present invention, the positive control sample contains any substance that has the same or similar structure as the misfolded protein and can compete with the microporous membrane for binding dyes (such as Congo red), such as a β-sheet structure Protein, specifically bovine serum albumin (BSA) after denaturation. In a specific embodiment of the present invention, the negative control sample is PBS buffer, and the positive control sample is PBS buffer containing misfolded BSA. In another specific embodiment of the present invention, the negative control sample is a urine sample that does not contain misfolded substances, and the positive control is a urine sample that contains misfolded proteins.

In some embodiments, the kit may also include the aforementioned comparison card, desiccant, and instructions. In a specific embodiment of the present invention, the sample is from the body fluid of a patient with a misfolded protein disease, including but not limited to whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid, pleural and ascites, tears, vaginal secretions, semen , Tissue lysate and its combination. When the sample contains blood and is colored, it can be centrifuged to remove red blood cells or other interference factors before testing. In a specific embodiment of the present invention, the misfolded protein disease is preeclampsia, and the sample is urine of pregnant women.

In order to make the technical problems, technical solutions, and beneficial effects solved by the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not to limit the present invention.

Example 1

This embodiment provides the type of capillary used in the present invention. As shown in Fig. 1A, the capillary tube used in the present invention can have various shapes (Fig. 1A). The cross section of the end in contact with the microporous membrane (that is, the end of the liquid outlet) can also be in various shapes, including but not limited to circles, ellipses, regular polygons, irregular polygons, and polygons with rounded corners (Figure 1B) . These capillaries can absorb 5-20μL of liquid. When the capillary contains liquid and one end of the liquid outlet is in contact with the filter paper, the rate of the liquid being released onto the filter paper is no more than 4μL/sec.

If you need to use multiple capillaries at the same time, for example, 3 capillaries can be made in-line. As shown in FIG. 1C, the capillary is connected by a connecting bridge 108, the liquid outlet 102 of the capillary 100 is used to suck liquid and contact the filter paper, and the other open port 106 penetrates the connecting bridge 108 and communicates with air.

Example 2

This embodiment provides a kit, which includes the devices shown in Figures 2, 3, and 4, and also includes one or two stainless steel capillaries with an inner diameter of 1.25 mm.

The device shown in FIG. 2 includes a test component 201, and the test component 201 includes filter paper 251. There are 3 ways to set the filter paper on the test part:

The first type: the test component 201 is a rectangular parallelepiped, and the upper surface of the rectangular parallelepiped is covered with a circular filter paper 251 (the setting method is shown in Fig. 4A, B).

The second type: the test component 201 is a rectangular parallelepiped, the upper surface of the rectangular parallelepiped is provided with a cylindrical groove 231, and the bottom of the groove is provided with filter paper 251 (as shown in Fig. 4C and D).

The third type: the test component 201 includes a cover plate 211 and a bottom plate 212, and a circular hole 271 is provided on the cover plate. When the cover plate 211 and the bottom plate 212 are glued, a layer of filter paper 251 is arranged in the middle (as shown in Figure 4E, F). In this way, after the cover plate 211 and the bottom plate 212 are glued, the circular hole forms a test slot 291, and the bottom of the test slot is the filter paper 251.

The device shown in Fig. 3 includes a test component 301, which is a rectangular parallelepiped. Two test grooves 311 and 312 are provided on one side surface. The test grooves 311 and 312 are each a cylindrical groove with a depth of approximately 2/3 of the thickness of the rectangular parallelepiped, filter paper is arranged in the groove, the filter paper is circular, slightly smaller than the cross section of the groove, and is laid flat on the bottom of the groove (the arrangement is shown in Figure 4C and D, and the filter paper is not shown in Figure 3) .

The specific detection steps used to detect whether the urine of pregnant women contains misfolded protein is to mix a sample taken from the urine of pregnant women with Congo red to obtain a mixed solution, and place the capillary vertically in the mixed solution for about 10 seconds (10μL can be drawn Left and right mixed liquid), transfer to the test tank, closely contact the outlet of the capillary with the filter paper, keep it upright, stay for about 15 seconds, the liquid in the capillary is slowly released onto the filter paper, remove the capillary, and observe the result.

The device shown in Figure 2 can only detect one sample to be tested. The device of FIG. 3 can detect two samples to be tested, or detect one sample to be tested and one control sample. The control sample can be a negative control sample or a positive control sample. The negative control sample is PBS buffer, and the positive control sample is PBS buffer containing misfolded BSA after special denaturation treatment.

Example 3

This embodiment provides a kit and a method of use. The kit includes the device shown in FIG. 5, FIG. 6, FIG. 7 or FIG. 8.

The detection device shown in FIG. 5 includes a test component 501 and a sample loading component 502. The test component 501 is composed of a first cover plate 511 and a first bottom plate 512. The first cover plate 511 is provided with three cylindrical grooves, which is the test Slots 514, 516, 518, and filter papers 534, 536, 538 are set at the bottom of the test slot according to Figure 4C and D. The lower surface of the first cover plate 511 is non-detachably connected with the upper surface of the first bottom plate 512 to form a whole. The sample loading component is composed of a second cover plate 521 and a second bottom plate 522. The second cover plate 521 is provided with three sample slots 525, 527, and 529. The lower surface of the second cover plate 521 is non-detachably connected with the upper surface of the second bottom plate 522 to form a whole; the side surface of the first cover plate 511 is directly connected to the side surface of the second cover plate 521 to form a whole; The side surface of 512 and the side surface of the second bottom plate 522 are directly connected to form a whole. As a result, the test component and the sample-loading component become a whole, and there is no physical difference.

The detection device shown in FIG. 6 includes a test component 601 and a sample loading component 602. The test component 601 is composed of a first cover plate 611 and a first bottom plate 612. The upper surface of the first cover plate 611 is provided with five test slots 631, 633, 635, 637, 639, filter paper 651, 653, 655, 657, 659 are respectively provided at the bottom of the test tank (the setting method is shown in Figure 4C and D). The sample loading component is composed of a second cover plate 621 and a second bottom plate 622. The upper surface of the second cover plate 621 is provided with five sample slots 641, 643, 645, 647, and 649. The side surface of the first cover plate 611 is connected to the side surface of the second cover plate 621 through the soft material 675, and the side surface of the first bottom plate 612 is not connected to the side surface of the second bottom plate 622. Therefore, the test component 601 and the sample loading component 602 can be folded together during transportation and storage, thereby saving space.

The detection device shown in Figure 7 includes a test component 701 and a sample loading component 702. The upper surface of the test component 701 is provided with three test slots 731, 733, 735, and the bottom of the test slot is provided with filter paper 751, 753, 755 (setting method As shown in Figure 4C and D). Three sample slots 741, 743, and 745 are provided on the upper surface of the sample loading part 702. The test component 701 and the sample loading component 702 are connected by a living hinge 705.

The detection device shown in Fig. 8 is basically the same as that shown in Fig. 7, except that the number of test slots and sample slots is more than that shown in Fig. 7 (14), which can be used to detect more samples.

Any kit also includes stainless steel capillaries with the same number of sample tanks and an inner diameter of 1.25 mm.

The specific detection procedure for detecting whether the urine of pregnant women contains misfolded protein is to mix a sample taken from the urine of pregnant women with Congo red (concentration 5mg/mL) in a sample tank at a ratio of 20:1 to obtain a mixed solution. Place it vertically in the mixed solution for about 10 seconds (approximately 10μL of mixed solution can be absorbed), transfer the capillary to the test tank, make the liquid outlet contact the filter paper, stay for about 15 seconds, the liquid in the capillary is slowly released onto the filter paper, Remove the capillary and observe the result.

The device shown in Figs. 5 and 7 can detect up to three samples to be tested. Of course, one sample can be tested, and the other two test slots are used to detect negative control samples and positive control samples. The detection method is the same. The device shown in Fig. 6 can detect 5 samples to be tested at most, or 3 samples to be tested, 2 control samples, ie, negative control samples and positive control samples. The device shown in Fig. 8 can detect up to 14 samples to be tested, or 12 samples to be tested, 1 negative control sample, and 1 positive control sample.

Example 4

This embodiment provides a kit and a method of use. The kit includes the device shown in FIG. 9, FIG. 10 or FIG. 11.

The detection device shown in FIG. 9 includes a test component 901, a sample loading component 902 and a sample spotting component 903. The test component 901 is composed of a first cover plate 911 and a first bottom plate (not shown in the figure), and 14 test slots 931 are provided on the first cover plate 911. The sample loading component 902 is composed of a second cover plate 921 and a second bottom plate (not shown in the figure), and 14 sample slots 941 are provided on the second cover plate 921. The first cover plate 911 and the first bottom plate are an integral body and are physically indistinguishable. The test slot 931 is provided with a filter paper 951, which is arranged as shown in FIG. 4C and D. The second cover plate 921 and the second bottom plate are integral and are physically indistinguishable. The test component 901 and the sample loading component 902 are connected together by a buckle 905, which can be separated and stacked together during transportation and storage, and connected together during use. The spotting component 903 is a hollow box-shaped component, which contains a capillary tube 933, which is a glass tube with an inner diameter of 1.2 mm, and the hollow box-shaped component is connected to the sample loading component 902 and the test component 901 by a buckle.

The detection device shown in FIG. 10 includes a test component 1001 , a sample loading component 1002, and a sample spotting component 1003. The test component 1001 is composed of a first cover plate 1011 and a first bottom plate 1012, and five test slots 1031 are provided on the first cover plate 1011. The sample loading component 1002 is composed of a second cover plate 1021 and a second bottom plate 1022. The second cover plate 1021 is provided with five sample slots 1041. The first cover plate 1011 and the first bottom plate 1012 are an integral body and are physically indistinguishable. The test slot 1031 is provided with filter paper 1051 in the manner shown in FIG. 4C and D. The second cover plate 1021 and the second bottom plate 1022 are integral and are physically indistinguishable. Similarly, the first cover plate 1011 and the second cover plate 1021 are integrated, and the first bottom plate 1012 and the second bottom plate 1022 are integrated. In other words, the test component 1001 and the sample loading component 1002 are integrated, and they are only differentiated in terms of division. The spotting component 1003 is a solid box-shaped component on which there are as many concave holes 1032 as the number of sample grooves. The diameter of the concave holes 1032 is slightly larger than the diameter of the capillary (not shown in the figure), and the depth is slightly larger than the length of the capillary. One capillary can be placed in each recessed hole. The upper surface of the solid box-shaped component is non-detachably connected with the lower surface of the first bottom plate 1012 and the bottom surface of the second bottom plate 1022 and forms a whole.

The detection device shown in FIG. 11 includes a test component 1101, a sample loading component 1102, and a sample spotting component 1103. The test component 1101 is composed of a first cover plate 1111 and a first bottom plate 1112. The first cover plate 1111 is provided with five test slots 1131; the sample loading component is composed of a second cover plate 1121 and a second bottom plate 1122. The second cover plate Five sample slots 1141 are provided on 1121. Like the detection device shown in FIG. 10, the detection device is a whole, except that it is divided into different parts, and the first cover 1111 is provided with a test slot 1131, and the filter paper is also placed according to the method of FIGS. 4C and D. The difference is that the spotting component 1103 is located on the side of the testing component 1101 and the sample loading component 1102. The spotting component 1103 is provided with a recessed hole 1151, which can accommodate not less than the number of capillaries 1153 (stainless steel capillaries with an inner diameter of 1.25 mm) of not less than the number of sample slots, and the depth is slightly greater than the length of the capillary 1153 (3 cm).

The use steps are the same as in Examples 2 and 3.

Example 5

This embodiment provides a kit and a method of use. The kit includes the devices shown in FIG. 12, FIG. 13 and FIG.

The detection device shown in FIG. 12 includes a test component 1201, a sample loading component 1202 and a sample spotting component 1203, and all components are made of PVC material. The test component 1201 is composed of a first cover plate 1211 and a first bottom plate (not shown in the figure). The sample loading component 1202 is composed of a second cover plate 1221 and a second bottom plate (not shown in the figure), and three sample slots 1241 are provided on the second cover plate 1221. The side surface of the first cover plate 1211 is non-detachably connected with the side surface of the second cover plate 1221 to form a whole, and a diversion groove 1206 is provided between the first cover plate 1211 and the second cover plate 1221, and the diversion groove 1206 is connected to the test The center line of the component 1201 and the carrier component 1202 is perpendicular, and the cross section is in the shape of an inverted triangle, which runs through the entire upper surface. The side surface of the first bottom plate is non-detachably connected with the side surface of the second bottom plate to form a whole. The first cover plate 1211 is provided with three round holes. When connecting with the first bottom plate, a layer of filter paper is placed in the middle (as shown in Figure 4E, F). In this way, the first cover plate 1211 and the first bottom plate are covered by four-corner snaps. Then, the circular hole forms a test slot 1231, and the bottom of the test slot 1231 is the filter paper 1251.

The spotting component 1203 is a spotting board. The side of the spotting board is provided with capillaries 1233. The number of capillaries is the same as the number of sample slots or the number of test slots 1231, that is, there are 3, and the arrangement is the same as that of the sample slot 1241 or the test slot 1231; The spotting plate is set on the same side of the capillary 1233. The two sides of the capillary 1233 also include positioning pins 1235. The first cover plate 1211 and the second cover plate 1221 are provided with positioning holes 1204 and 1208. When the positioning pin 1235 is inserted into the positioning hole 1204 or 1208 In the middle, the capillary 1233 can be inserted into the sample slot 1241 or the test slot 1231. The sample loading component 1202 is also provided with concave holes (not shown in the figure) on the side of the test component 1201. The arrangement of the concave holes is the same as the arrangement of the capillary 1233 and the positioning pin 1235 on the spotting board, so that the spotting component 1203 can be fixed on The sample loading part 1202 is on. A protrusion 1237 is also provided on the spot template to facilitate the insertion and removal of the spot template.

In the sample tank 1241, add Congo red in advance, one of the sample tanks 1241 is also filled with a positive control sample (PBS buffer containing BSA), and the other sample tank is filled with a negative control sample (PBS buffer without BSA), and Seal with tin foil.

The detection device shown in Figure 13 is basically the same as the detection device shown in Figure 12, except that the number of test slots and sample slots is more than that shown in Figure 12 (5), which can be used to detect more samples .

The method of using this kit is as follows: 1. Pull out the spotting parts and place the main body of the detection device (packaging test parts and sample loading parts) horizontally on the table; 2. Use the positioning needle to pierce the tin foil on the sample tank in turn (Figure 14A); 3. Add pregnant woman's urine to the sample tank to be tested; 4. Insert the positioning pin on the spotting component into the positioning hole of the second cover (Figure 14B), thereby spotting the sample component Insert the capillary tube of the sample into the liquid in the sample tank, so that the liquid will automatically rise in the capillary; 5. Insert the positioning pin of the spotting part into the positioning hole of the first cover (Figure 14C), so that the spot on the spotting part The capillary is in direct contact with the microporous membrane in the test tank (shown in the cross-sectional view of Figure 14D), and let it stand for 5-10 seconds; 6. Remove the spotting parts and observe the test results. 7. If the result is shown in Figure 15A, that is, the spotted part shows more concentrated and non-diffuse red spots (Figure 15A, a), or in addition to the spotted parts that show more concentrated and non-diffused red spots, there are "pseudopods "(Figure 15A, b), or accompanied by a slight diffusion (Figure 15A, c), the pregnant urine sample is judged as negative, that is, it does not contain misfolded proteins or contains misfolded proteins below the reference value.

If the result is shown in Figure 15B, that is, the spotted part shows smaller red spots, but there is more obvious diffusion around (Figure 15B, d), or the spotted spot shows red spots, with irregularly spreading light perfect circles ( Figure 15B, e), or the formation of uniform and larger diffuse spots (Figure 15B, f), the pregnant woman's urine sample is judged to be positive, that is, it contains misfolded protein or contains a misfolded protein higher than the reference value.

The detection device provided in this embodiment integrates equipment and reagents, which greatly compresses the packaging space, facilitates transportation and storage, and facilitates laboratory operations.

Example 6

This embodiment provides a simple kit and a method of use. The kit includes a filter paper and a capillary tube (a stainless steel capillary tube with an inner diameter of 1.25 mm). The usage method is as follows: 1. Place the filter paper horizontally on the table; 2. Mix the urine sample taken from the pregnant woman with the Congo red dye into a mixed solution; 3. Take the capillary to absorb the mixed solution and transfer it to the filter paper for close contact and close contact , Stay for more than 5 seconds to make the liquid in the capillary be slowly and fully released on the filter paper; 4. Leave it for about 10 seconds to observe the test results.

Fig. 16 shows a schematic diagram of the detection process of the detection device of the present invention. After the capillary contacts the filter paper, the sample is slowly released on the filter paper and spreads outward at the contact position to form stained spots of different sizes.

Example 7

In order to compare the influence of capillary and micropipette or dropper on the detection results, the inventor designed the following experiment:

1. The capillary used in the capillary, micropipette and dropper is a glass capillary with a circular outlet of 1.25 mm in inner diameter and 10 cm in length. The capillary is marked with a 10μL graduation line. The micropipette used is a 10 μL micropipette. The dropper used is 100 μL, and each test draws 10 μL of liquid.

2. Negative and positive samples Negative samples are urine samples of pregnant women judged by doctors as non-preeclampsia based on clinical results, and positive samples are urine samples of pregnant women judged as preeclampsia by doctors based on clinical results.

3. Detection

Congo red was added to the negative and positive samples at a ratio of 50:1. Insert the capillary into the negative sample or the positive sample respectively. When the liquid rises to the 10 μL mark, take out the capillary and touch the end of the liquid to the filter paper. After the sample is absorbed by the filter paper, remove the capillary and observe after 15 seconds of diffusion. Test results.

Similarly, use a micropipette and a dropper to draw 10 μL of negative or positive samples, and then contact the tip of the micropipette or the dropper with the filter paper to release the absorbed liquid onto the filter paper. After a period of diffusion, observe the test results.

In order to compare the difference between the contact spotting and the hanging spotting on the detection effect, the capillary, micropipette and dropper were used to draw 10 μL of negative or positive samples, and the capillary outlet, micropipette tip or dropper was suspended in the air. At 0.5-2cm above the filter paper, release the absorbed mixed liquid onto the filter paper, and after spreading for a period of time, observe the test results.

Measure the radius of the spread spot of the negative sample and the positive sample. The spread spot radius is the length from the center of the spot to the farthest position of the spot. And calculate the radius ratio of the diffusion spot radius of the positive sample to the diffusion radius of the negative sample. Each experiment was repeated 5 times.

4. Results

Figure 17 or Table 1 shows the spread of spots using capillary, micropipette, and dropper. For positive samples:

Regardless of the spotting device and the spotting method, the positive samples will form uniform and larger diffusion spots, with almost no difference.

For negative samples:

When the three kinds of spotting devices (capillary tube, micropipette and dropper) are suspended in the air, the negative samples form relatively large spots. The radius ratio of the spots formed by the same spotting method as the positive samples is about 1.2, which is relatively small. , Easy to form misjudgment.

When the spot is contacted, the spots formed by the capillary detection are more concentrated, do not diffuse outward or rarely diffuse, and the radius ratio of the spot formed by the positive sample is more than 2.5. The micropipette and the dropper are in contact with the spot, and the diffusion spots formed by the negative samples are relatively large and irregularly diffuse outward, with a radius ratio of about 1.6.

Table 1 The effect of spotting with capillary, micropipette and dropper on the spread of spots (n=5, p＜0.01)

In summary, the use of capillary contact spot detection is the best, the distinction is more obvious, it is not easy to cause misjudgments, and the results are intuitive and reliable.

Example 8

In order to compare the influence of different inner diameters of capillary tubes on the detection results, the inventor designed the following experiment:

1. Capillary

The capillaries used are stainless steel capillaries with inner diameters of 0.4, 0.5, 0.6, 0.7, 0.9, 1.12, 1.25, 1.45, 1.69, 1.99, 2.4, 2.64, and 2.8 mm and a length of 3 cm. The capillaries are marked with a 10 μL scale. line.

2. Negative and positive samples

Negative samples are urine samples of pregnant women judged by doctors as non-preeclampsia based on clinical results, and positive samples are urine samples of pregnant women judged by doctors as preeclampsia based on clinical results.

3. Detection

Congo red was added to the negative and positive samples respectively. Insert capillary tubes with different inner diameters into negative samples or positive samples respectively, and stay for more than 30 seconds. When the liquid no longer rises, take out the capillary and touch the end of the liquid to the filter paper until all the samples are absorbed by the filter paper (about 3-10 seconds ), remove the capillary, after a period of diffusion, observe the test results.

Measure the radius of the spread spot of the negative sample and the positive sample. The spread spot radius is the length from the center of the spot to the farthest position of the spot. And calculate the radius ratio of the diffusion spot radius of the positive sample to the diffusion radius of the negative sample. Each experiment was repeated 5 times.

4. Results

The spot diffusion of spotting with different inner diameter capillaries is shown in Figure 18 or Table 2. It can be seen that when the inner diameter of the capillary is too small (for example, less than 0.7mm), the speed of the liquid from the capillary to the filter paper is too slow, and the positive sample The spot spread radius is too small, and it is not much different from the spot spread radius of the negative sample. As the inner diameter of the capillary increases, the spread of the spots of the negative samples does not increase significantly, but the spread of the spots of the positive samples increases rapidly. When the inner diameter of the capillary increases to a certain extent (for example, greater than 3mm), because the contact area between the liquid and the filter paper is too large, the spot diffusion radius of the negative sample also begins to increase, but the spot diffusion radius of the positive sample does not change significantly, resulting in a radius ratio No more increase or even a downward trend. When the capillary increases further, the liquid can no longer enter the capillary spontaneously, making detection impossible. In this test, the detection effect is better when the inner diameter of the capillary is 0.7-2.8mm, and the detection effect is better when the inner diameter of the capillary is 1.12-2.64mm.

Table 2 The influence of different capillary inner diameters on spot diffusion (n=5, p＜0.01)

Example 9

In order to compare the influence of different spot sizes on the test results, the inventor designed the following experiment:

1. Capillary

The capillary used is a glass capillary with an inner diameter of 1.25 mm and a length of 5 cm. The capillary has a maximum volume of 20 μL. The capillary is marked with a scale line with a range of 1-20 μL and an accuracy of 1 μL.

2. Negative and positive samples Negative samples are urine samples of pregnant women judged by doctors as non-preeclampsia based on clinical results, and positive samples are urine samples of pregnant women judged as preeclampsia by doctors based on clinical results.

3. Detection

Congo red was added to the negative and positive samples respectively. Insert the capillary into the negative sample or the positive sample, and draw 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 μL of liquid, and then remove the capillary. Touch the end of the liquid absorbing liquid to the filter paper. After the sample is completely absorbed by the filter paper, remove the capillary, and after diffusion for a period of time, observe the detection result.

Measure the radius of the spread spot of the negative sample and the positive sample. The spread spot radius is the length from the center of the spot to the farthest position of the spot. And calculate the radius ratio of the diffusion spot radius of the positive sample to the diffusion radius of the negative sample. Each experiment was repeated 5 times.

4. Results

The spread of spots with different spot sizes is shown in Figure 19 or Table 3. It can be seen that with the increase of spot size, the spot spread radius of negative samples does not increase significantly, but the spot spread radius of positive samples increases rapidly. Negative samples and positive samples The distinction between samples is becoming more and more obvious, manifested as the radius ratio gradually increases. But when the spot volume reaches 16μL, the spot diffusion radius of the positive sample increases slowly, or even no longer. This is affected by the diffusion mechanics, making the spot diffusion concentrated in a certain area. On the other hand, as the amount of sample increases, the capillary adsorption time becomes longer and longer, and because the negative sample does not diffuse or diffuses to a small degree, it is difficult for the liquid in the capillary to be completely absorbed to the filter paper or it takes longer It is completely adsorbed, resulting in too long detection time. Comprehensive consideration, the spot size is at least 4μL, and the effect is better at 5-15μL.

Table 3 The influence of different spot size on spot spread (n=5, p＜0.01)

The above examples are used here to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the technology disclosed in the above examples represents the technology discovered by the inventor that can be used to implement the present invention, and therefore can be regarded as a preferred solution for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, and the same or similar results can still be obtained without departing from the spirit or scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The public quotations herein and the materials cited by them will be incorporated by reference. .

Those skilled in the art will be aware of, or through routine experimentation, many equivalent techniques for the specific embodiments of the invention described herein. These equivalents will be included in the scope of the patent application.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the patent application scope of this application.

100‧‧‧Capillary 102‧‧‧Outlet 106‧‧‧Open port 108‧‧‧Connecting bridge 160A‧‧‧Combination 162‧‧Capillary 164‧‧‧Outlet 166‧‧‧Nozzle 168‧ ‧‧Microporous membrane 180‧‧‧Connecting bridge 182‧‧‧Capillary 184‧‧‧Liquid outlet 186‧‧‧Tube 188‧‧Microporous membrane 192‧‧Capillary 194‧‧‧Liquid outlet 196‧ ‧‧Nozzle 198‧‧‧Microporous membrane 201‧‧‧Test part 211‧‧‧Cover plate 212‧‧‧Bottom plate 231‧‧‧Groove 251‧‧‧Filter paper 271‧‧‧Circular hole 291‧‧‧Test Slot 301‧‧‧Test component 311‧‧‧Test slot 312‧‧‧Test slot 501‧‧‧Test component 502‧‧‧Sample loading component 511‧‧‧First cover 512‧‧‧First bottom 514‧‧ ‧Test slot 516‧‧‧Test slot 518‧‧‧Test slot 521‧‧‧Second cover plate 522‧‧‧Second bottom plate 525‧‧‧Sample slot 527‧‧‧Sample slot 529‧‧‧Sample slot 534‧ ‧‧Filter paper 536‧‧‧Filter paper 538‧‧‧Filter paper 601‧‧‧Test component 602‧‧‧Sample loading component 611‧‧‧First cover 612‧‧‧First bottom plate 621‧‧‧Second cover 622 ‧‧‧Second bottom plate 631‧‧‧Test slot 633‧‧‧Test slot 635‧‧‧Test slot 637‧‧‧Test slot 639‧‧‧Test slot 641‧‧‧Sample slot 643‧‧‧Sample slot 645‧ ‧‧Sample tank 647‧‧‧Sample tank 649‧‧‧Sample tank 651‧‧‧Filter paper 653‧‧‧Filter paper 655‧‧‧Filter paper 657‧‧‧Filter paper 659‧‧‧Filter paper 675‧‧‧Soft material 701‧‧ ‧Test component 702‧‧‧Sample loading component 705‧‧‧Living hinge 731‧‧‧Test slot 733‧‧‧Test slot 735‧‧‧Test slot 741‧‧‧Sample slot 743‧‧‧Sample slot 745‧‧‧ Sample slot 751‧‧‧Filter paper 753‧‧‧Filter paper 755‧‧‧Filter paper 901‧‧Test part 902‧‧‧Sample loading part 903‧‧‧Sampling part 905‧‧‧Snap 911‧‧‧First cover Plate 921‧‧‧Second cover plate 931‧‧‧Test slot 933‧‧‧Capillary tube 941‧‧‧Sample slot 951‧‧‧Filter paper 1001‧‧‧Test part 1002‧‧‧Sample loading part 1003‧‧‧Spotting Part 1011‧‧‧First cover 1012‧‧‧First bottom 1021‧‧‧Second cover 1022‧‧‧Second bottom 1031‧‧‧Test slot 1032‧‧‧Concave hole 1041‧‧‧Sample slot 1051 ‧‧‧Filter paper 1101‧‧‧Test component 1102‧‧‧Sample loading component 1103‧‧‧Sampling component 1111‧‧‧First cover 1112‧‧‧First bottom plate 1121‧‧‧Second cover 1122‧‧ ‧Second bottom plate 1131‧‧‧Test slot 1141‧‧‧Sample slot 11 51‧‧‧Concave hole 1153‧‧‧Capillary tube 1201‧‧‧Test component 1202‧‧‧Sample loading component 1203‧‧‧Spotting component 1204‧‧Locating hole 1206‧‧‧Diversion groove 1208‧‧‧Locating hole 1211‧‧‧First cover 1221‧‧‧Second cover 1231‧‧‧Test slot 1233‧‧‧Capillary 1235‧‧‧Locating pin 1237‧‧‧Protrusion 1241‧‧‧Sample slot 1251‧‧‧Filter paper

Figure 1A shows part of the capillary types used in the present invention (A: capillary shape, from left to right: upper and lower thick capillary, upper thick and lower thin capillary, segmented thick capillary, segmented upper thick and lower thin capillary, Wave-shaped capillary, pentagonal cylindrical capillary, pentagonal star capillary), Figure 1B shows other shapes that can be selected for the cross section of the end of the capillary in contact with the microporous membrane, and Figure 1C shows a schematic diagram of three capillaries made into a row capillary. The left side of the figure is a front view, the upper right is a top view, and the lower one is a left view. Figure 2 shows a schematic diagram of a specific structure of the test component of the present invention. Figure 3 shows a schematic diagram of another specific structure of the test component of the present invention. Fig. 4 shows the arrangement of the microporous membrane 4, A, C, and E are respectively schematic diagrams of the arrangement, and B, D, and F are corresponding cross-sectional views respectively. Figure 5 shows a schematic diagram of a specific structure of the detection device of the present invention (A: top view, B: front view, C: left view). Figure 6 shows a schematic diagram of another specific structure of the detection device of the present invention (A: top view, B: front view, C: left view when unfolded, D: left view when folded). Fig. 7 shows a schematic diagram of another specific structure of the detection device of the present invention (A: top view, B: bottom view). Figure 8 shows a schematic diagram of another specific structure of the detection device of the present invention. Fig. 9 shows a schematic diagram of another specific structure of the detection device of the present invention. Figure 10 shows a schematic diagram of another specific structure of the detection device of the present invention (A: top view, B: front view, C: left view). Figure 11 shows a schematic diagram of another specific structure of the detection device of the present invention (A: top view, B: front view, C: left view). Fig. 12 shows a schematic diagram of another specific structure of the detection device of the present invention. Fig. 13 shows a schematic diagram of another specific structure of the detection device of the present invention. Fig. 14 shows a schematic diagram of a method of using a specific structure of the detection device of the present invention. Figure 15 shows the detection result of the detection device or kit of the present invention. Fig. 16 shows a schematic diagram of the present invention using a row of capillaries for detection. Figure 17 shows the effect of spotting using capillaries, micropipettes and droppers on spot spread. Figure 18 shows the effect of different capillary inner diameters on spot spread. Figure 19 shows the effect of different spot sizes on spot spread. Figure 20 shows a schematic diagram of the automatic determination system of the present invention.

1201‧‧‧Test parts

1202‧‧‧Sample loading parts

1203‧‧‧Sampling parts

1204‧‧‧Locating hole

1206‧‧‧Diversion groove

1208‧‧‧Locating hole

1211‧‧‧First cover

1221‧‧‧Second cover

1231‧‧‧Test slot

1233‧‧‧Capillary tube

1235‧‧‧Locating pin

1237‧‧‧Protrusion

1241‧‧‧Sample tank

1251‧‧‧Filter paper

Claims (10)

A device for detecting whether a sample contains misfolded proteins, including: a liquid outlet with a cross-sectional area of no more than 9 mm ² and a capillary capable of sucking and holding at least 5 μL of liquid; and a microcapillary capable of competing with misfolded proteins for binding dye A porous membrane, wherein the liquid outlet of the capillary is in contact with and closely adhered to the surface of the microporous membrane. The device according to claim 1, wherein the capillary tube contains at least 5 μL of a mixed solution formed by mixing the dye and the biological sample, wherein the dye is selected from the group consisting of heterocyclic dyes, Congo red, Thioflavin, and Evans Blue One or more of. The device according to claim 2, wherein the surface of the microporous membrane around the capillary liquid outlet is stained due to the slow release of the mixed liquid in the capillary from the capillary liquid outlet. A device for detecting whether a sample contains misfolded proteins, comprising: a panel with at least a first row of grooves and a second row of grooves, and a spotting part, wherein the first row of grooves includes an adjacent one The sample tank to be tested, a negative control sample tank, and a positive control sample tank are respectively filled with 30-500μL or more of liquid; the second row of grooves includes at least three bottoms covered with misfolded proteins that can compete for binding Dye microporous membrane test tank; the spotting component contains at least three parallel liquid outlets of the same length, the cross-sectional area of which is not more than 9mm ² and the capillaries capable of sucking and accommodating at least 5μL of liquid are fixed side by side on a beam And the distance between the capillaries corresponds to the distance between the sample grooves and the distance between the test grooves such that the at least three capillaries are inserted into the corresponding sample groove or the corresponding test groove at the same time. The device according to claim 4, wherein the sample tank to be tested contains a dye or dye solution that can specifically bind to misfolded proteins; the negative control sample tank contains the negative pair According to the sample; the positive control sample slot is equipped with the positive control sample; the sample slot is sealed with a sealing material. A kit for detecting whether a sample contains misfolded protein, including a capillary with a cross-sectional area of the liquid outlet not exceeding 9 mm ² and capable of sucking and holding at least 5 μL of liquid; and a microporous membrane that can compete with the misfolded protein for binding dye. The kit according to claim 6, further comprising a comparison card printed with examples of at least negative and positive sample spread results. A kit for detecting whether a sample contains misfolded protein, including the device of claim 4 or 5. A detection system for detecting whether a sample contains misfolded proteins, including: the device of claim 4 or 5; the signal acquisition module includes an optical signal collector; and an information memory for storing the process collected by the signal acquisition module Processed or unprocessed signal. A method for detecting whether a sample contains misfolded protein, including the following steps: mixing the sample with a dye that can bind to the microporous membrane and misfolded protein to form a mixed solution; the cross-sectional area of the liquid outlet does not exceed 9mm ² and can be The capillary tube for sucking and holding at least 5 μL of the mixed solution sucks at least 4 μL of the mixed solution; the liquid outlet of the capillary is in close contact with the microporous membrane to slowly release the mixed solution in the capillary To the microporous membrane; based on the color of the dye, observe the diffusion of the mixed solution on the microporous membrane to determine whether the sample contains misfolded proteins.

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