WO2019128506A1 - 一种检测错误折叠蛋白质的装置、试剂盒和方法 - Google Patents

一种检测错误折叠蛋白质的装置、试剂盒和方法 Download PDF

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Publication number
WO2019128506A1
WO2019128506A1 PCT/CN2018/115444 CN2018115444W WO2019128506A1 WO 2019128506 A1 WO2019128506 A1 WO 2019128506A1 CN 2018115444 W CN2018115444 W CN 2018115444W WO 2019128506 A1 WO2019128506 A1 WO 2019128506A1
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Prior art keywords
sample
capillary
microporous membrane
dye
liquid
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PCT/CN2018/115444
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English (en)
French (fr)
Inventor
李兴民
徐军
汤小彬
张以哲
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浙江数问生物技术有限公司
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Application filed by 浙江数问生物技术有限公司 filed Critical 浙江数问生物技术有限公司
Priority to RU2020124438A priority Critical patent/RU2020124438A/ru
Priority to EP18894064.7A priority patent/EP3734282A4/en
Priority to US16/958,652 priority patent/US20210156853A1/en
Priority to BR112020013608-5A priority patent/BR112020013608A2/pt
Publication of WO2019128506A1 publication Critical patent/WO2019128506A1/zh
Priority to ZA2020/03555A priority patent/ZA202003555B/en
Priority to PH12020550962A priority patent/PH12020550962A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Definitions

  • the present invention relates to a detection device and a detection method, and in particular to an apparatus and method for detecting misfolded proteins in a biological sample.
  • Misfolded proteins are proteins formed by misfolding, which are different from the conformation of proteins formed by natural folding.
  • Protein misfolding forms a large number of beta-sheet structures arranged in opposite directions. The structure is highly exposed due to hydrophobic surface exposure, resulting in a high degree of adhesion between protein molecules.
  • the ⁇ -Sheet structure of other misfolded proteins is aggregated, and the protein molecules that are aggregated together act as crystal nuclei to make other unrelated proteins. Polymerization forms aggregates of misfolded proteins that can accommodate virtually unlimited polypeptide chains (Misfolded Protein Aggregates: Mechanisms, Structures and Potential for Disease Transmission, Semin Cell Dev Biol. 2011 July; 22(5): 482-487.) Polymers and fibrous aggregates.
  • a first aspect of the present invention provides an apparatus for detecting whether a sample contains a misfolded protein, comprising a test part and a spotting part, the test part comprising a microporous film, and the spotting part comprising 1 , 2, 3 or more capillaries.
  • the test component includes a first cover plate and a first bottom plate, the first cover plate comprising a microporous film.
  • the apparatus further includes a sample loading component having one or more sample reservoirs thereon for containing the sample (test sample, negative control sample, and/or positive control sample) and/or dye.
  • the first cover is covered with a microporous membrane.
  • a test slot is provided on the first cover, and the test slot contains a microporous film.
  • the spotting member is a hollow box-like member containing 1, 2, 3 or more capillaries; optionally, the spotting member is a solid box-like member provided thereon 1, 2, 3 or more recessed holes for placing respective 1, 2, 3 or more capillaries; optionally, the spotting member is a spotting plate comprising a body portion and a capillary tube, the body portion being A plate-like member having capillary tubes disposed on the side, and the number of capillaries is 1, 2, 3 or more.
  • One end of the capillary is a liquid withdrawal end for aspirating and releasing the liquid, and the other end is connected to the sample plate body; optionally, the spotting member is a capillary.
  • a second aspect of the invention provides a kit for detecting whether a misfolded protein is contained in a sample, comprising the device of the first aspect of the invention, further comprising a dye capable of binding a microporous membrane and a misfolded protein, preferably a dye One or more selected from the group consisting of heterocyclic dyes, Congo red, thioflavin, and Evans blue, and more preferably, the dye is Congo red.
  • a third aspect of the invention provides a kit for detecting whether a sample contains misfolded proteins, comprising a microporous membrane and 1, 2, 3 or more capillaries.
  • a dye capable of binding a microporous membrane and a misfolded protein is further included, and preferably, the dye is selected from one or more of a heterocyclic dye, Congo red, thioflavin, and Evans blue, and more Preferably, the dye is Congo red.
  • the above kit further includes a control sample including a negative control sample and/or a positive control sample.
  • a fourth aspect of the invention provides a method of detecting whether a sample contains misfolded proteins, comprising the steps of:
  • the mixed solution is siphoned through the capillary without external force entering the capillary.
  • Preferably 5 to 25 ⁇ L, more preferably 8 to 15 ⁇ L of the mixture is drawn into the capillary and completely released into the microporous membrane.
  • a fifth aspect of the invention provides a combination for detecting whether a sample contains misfolded proteins, comprising a capillary tube and a microporous membrane, wherein a liquid outlet of the capillary is in contact with and adheres to a surface of the microporous membrane.
  • the capillary contains a mixture of a dye and a sample mixed with the microporous membrane and the misfolded protein, and the liquid outlet of the capillary is in close contact with the microporous membrane and is in close contact with each other, preferably,
  • the dye is selected from one or more of a heterocyclic dye, Congo red, thioflavin, and Evans blue, more preferably Congo red, and a certain amount means 5 ⁇ L or more, preferably 5 to 25 ⁇ L, more preferably 8-15 ⁇ L. .
  • Figure 1A shows a part of the capillary type used in the present invention (A: capillary shape, from left to right: upper and lower equal thick capillary, upper thick lower capillary, segmented equal thick capillary, segmented upper and lower capillary, Wave-shaped capillary, pentagonal capillary, pentagonal capillary),
  • Figure 1B shows other shapes that can be selected from the cross-section of the capillary in contact with the microporous membrane.
  • Figure 1C shows a schematic diagram of three capillary tubes made up of a tandem capillary. The left side of the figure is the main view, the top right is the top view, and the lower figure is the left view.
  • Fig. 2 is a schematic view showing a specific structure of a test member of the present invention.
  • Fig. 3 is a schematic view showing still another specific structure of the test member of the present invention.
  • Fig. 4 shows the arrangement of the microporous film 4
  • A, C, and E are schematic views, respectively
  • B, D, and F are corresponding cross-sectional views, respectively.
  • Fig. 5 is a view showing a specific structure of the detecting device of the present invention (A: top view, B: front view, C: left view).
  • Fig. 6 is a view showing another specific structure of the detecting device of the present invention (A: top view, B: front view, C: left view when unfolded, D: left view when folded).
  • Fig. 7 is a view showing another specific structure of the detecting device of the present invention (A: top view, B: bottom view).
  • Fig. 8 is a view showing another specific structure of the detecting device of the present invention.
  • Fig. 9 is a view showing another specific structure of the detecting device of the present invention.
  • Fig. 10 is a view showing another specific structure of the detecting device of the present invention (A: top view, B: front view, C: left view).
  • Fig. 11 is a view showing another specific structure of the detecting device of the present invention (A: top view, B: front view, C: left view).
  • Fig. 12 is a view showing another specific structure of the detecting device of the present invention.
  • Fig. 13 is a view showing another specific structure of the detecting device of the present invention.
  • Fig. 14 is a view showing the use of a specific structure of the detecting device of the present invention.
  • Figure 15 shows the detection results of the detection device or kit of the present invention.
  • Figure 16 is a schematic view showing the detection of the present invention using a tandem capillary.
  • Figure 17 shows the effect of spotting on a spot using capillary, pipette and dropper.
  • Figure 18 shows the effect of different capillary inner diameters on spot spread.
  • Figure 19 shows the effect of different spotting on spot spread.
  • Figure 20 is a diagram showing the automatic determination system of the present invention.
  • misfolded protein or “misfolded protein” described herein is relative to a correctly folded protein. Protein misfolding forms a large number of inverted ⁇ -sheet structures that result in a high degree of adhesion between protein molecules due to hydrophobic surface exposure, and other misfolded proteins.
  • the ⁇ -Sheet structure naturally aggregates, and the protein molecules that are brought together act as crystal nuclei to copolymerize other unrelated misfolded proteins to form aggregates (oligomers and fibrous aggregates) of misfolded proteins.
  • the meaning of "misfolded protein” includes these aggregates formed by misfolded proteins.
  • ⁇ -sheet structure can be formed, and a ⁇ -sheet structure of different protein sources can be formed. It can interact to form misfolded protein aggregates containing different proteins that can conform specifically to Congo red.
  • microporous membrane refers to a membrane made of a microporous material or a surface covering microporous material, which has such a function due to its spatial structure and/or compositional specificity: Ability to compete with misfolded proteins for binding dyes, ie 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 along with the solvent on the microporous membrane, forming smaller colored spots If the dye binds to the misfolded protein, it does not bind to the microporous material, so the dye and misfolded protein binding product can diffuse on the microporous membrane, resulting in larger colored spots.
  • the microporous material can be any material known to those skilled in the art that contains a large amount of free hydroxyl groups, such as cellulose, and the microporous membrane is a cellulose membrane, such as a filter paper.
  • the dye may be of any of the above characteristics well known to those skilled in the art and capable of conformationally conforming to the misfolded protein conformation, such as Congo red.
  • the “capillary” according to the present invention is a hollow tube indicating that the inner diameter of the liquid port does not exceed 3.5 mm or the cross-sectional area of the liquid outlet is not more than 9 mm 2 , and when the capillary is inserted into the liquid, the liquid can infiltrate the inner surface of the capillary, and the performance If the liquid level in the capillary is higher than the external liquid level, that is, a "capillary phenomenon" occurs, when the capillary leaves the liquid, the liquid remaining inside the capillary due to the surface tension is not less than 5 ⁇ L, preferably, the inner surface of the capillary is hydrophilic. Sex.
  • the material and texture or treatment of the capillary can be a capillary known to those skilled in the relevant art to achieve the volume of the immersion liquid described above.
  • the diffusion area of a dye capable of specifically binding to a misfolded protein conformation such as a Congo red solution
  • a microporous membrane such as a filter paper
  • a pipette is used for pipetting (such as a pipette, a pipette, etc.)
  • the droplets are directly dropped on the filter paper, forming a large contact surface with the filter paper and rapidly spreading on the filter paper, even if Negative samples without misfolded proteins also showed large color spots on the filter paper, which were often not significantly different from the color spots of the positive samples, resulting in inaccurate detection results, resulting in many false positives and false negatives.
  • the suction port of the pipette or pipette used is small, this defect cannot be overcome.
  • the inventors have further found that if a capillary spot (rather than a drop) is used, a sufficient volume of the mixture of the dye and the sample is sucked by the capillary so that the outlet of the capillary is in close contact with the filter paper, so that the mixture in the capillary is affected by the filter paper.
  • the water absorption of the material is slowly released onto the filter paper, which can significantly increase the difference of the color spots generated by the negative-positive sample, greatly reduce the false positive rate, and significantly improve the detection accuracy.
  • the present invention provides a method of detecting whether a sample contains misfolded proteins or misfolded protein aggregates, including the following steps:
  • the sample contains misfolded protein or misfolded protein aggregates, preferably, when the diffusion area exceeds the reference value, the determination sample contains Misfolded proteins or misfolded protein aggregates.
  • the present invention provides an assembly or device for detecting whether a sample contains misfolded protein aggregates, which can be used to carry out the above detection method.
  • the assembly comprises a capillary and a microporous membrane, wherein the outlet of the capillary contacts and is in close contact with the surface of the microporous membrane.
  • the methods and combinatorials of the invention can be used to detect a variety of samples, such as whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid, pleural fluid, tears, vaginal secretions, semen, tissue lysates And combinations thereof, when the sample contains blood and is colored, it can be centrifuged to remove red blood cells or other interfering factors before testing.
  • the methods and compositions of the present invention are particularly useful for detecting whether a pregnant woman's urine contains misfolded proteins or misfolded protein aggregates, thereby predicting, detecting, screening or diagnosing whether a pregnant woman has pre-eclampsia or eclampsia Early risk.
  • the microporous film in the present invention may be a film made of a material containing a free hydroxyl group and having water absorbability.
  • the microporous membrane is a cellulose membrane made of cellulose containing free hydroxyl groups, such as filter paper, writing paper, printing paper, label paper.
  • the dye may be any dye capable of conformationally binding to the misfolded protein, and such dye should also competitively bind to the material in the microporous membrane (e.g., cellulose containing free hydroxyl groups).
  • the dye is Congo red.
  • the dye is thioflavin or Evans blue.
  • the dye may be a solid dye directly mixed with a sample such as urine and dissolved in a liquid sample, or may be mixed with a sample such as urine in the form of a solution, and the concentration of the dye such as Congo red may be 0.01 to 2 mg/mL in the resulting mixture. It is preferably 0.02 to 1 mg/mL, more preferably 0.05 to 0.5 mg/mL.
  • the capillary has two ends along the length direction of the tube, one end is a liquid outlet, the other end may be open or closed, and the inner diameter of the outlet of the capillary is less than or equal to 3.5 mm or the horizontal of the liquid outlet.
  • the area of the section does not exceed 9 mm 2 .
  • the capillary should have sufficient tube length to draw and contain at least 4 ⁇ L of the mixture, preferably at least 5 ⁇ L of the mixture, at least 8 ⁇ L of the mixture, more preferably from 8 to 15 ⁇ L.
  • the capillary is ingested in an amount of 2 to 30 ⁇ L, 4 to 30 ⁇ L, more preferably 5 to 25 ⁇ L, 5 to 20 ⁇ L, 4 to 17 ⁇ L, or 5 to 17 ⁇ L, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ⁇ L; more preferably 8 to 16 ⁇ L.
  • the capillary is substantially uniform in thickness along the length of the tube, and the cross section including the outlet opening may be circular or any other regular or irregular shape, such as those shown in FIG.
  • the liquid outlet has an inner diameter of about 0.5 to 3 mm, 0.7 to 3 mm, preferably 0.9 to 2.8 mm, or a cross-sectional area of about 0.2 to 7 mm 2 , preferably about 0.64 to 6.2 mm 2 .
  • the capillary draw mixture draws at least 5 [mu]L of liquid by capillary action.
  • the water absorption of the liquid by the microporous membrane is passively released from the capillary into the microporous membrane upon contact of the liquid outlet with the microporous membrane.
  • the rate at which the liquid is released into the microporous membrane is no more than 4.5 ⁇ L/sec, no more than 4 ⁇ L/sec, preferably 0.5-4 ⁇ L/sec, for example 0.5, 1, 1.5, 2, 2.5, 3, 3.5. Or 4 ⁇ L/sec, more preferably 1 to 3 ⁇ L/sec, such as 1.2, 1.6, 1.7, 1.8, 2.3, 2.6 or 2.8 ⁇ L/sec.
  • the results obtained by the method, assembly, device or kit of the present invention are negative, that is, the mixture of the sample and the dye is sampled into the micropores by capillary tubes.
  • the diffusion radius is less than a reference value, which may be clinically determined by a certain number (eg, 50-100). The maximum value of the radius of the diffusion spot formed by the sample of the patient determined to be negative is determined.
  • the results obtained by the method, assembly, device or kit of the present invention are positive, that is, the mixture of the sample and the dye is transferred to the microporous membrane by a capillary tube. Thereafter, a diffusion spot larger than the negative sample is formed on the microporous membrane.
  • the positive sample produces a larger diffusion spot with a diffusion radius greater than or equal to a particular reference value, and the reference value can be diffused by a certain number (eg, 50-100) of positive samples. The minimum of the radius of the spot is determined.
  • determining whether the sample contains misfolded protein or misfolded protein aggregates comprises comparing the diffusion result of the mixture on the microporous membrane to a comparison card comprising at least a negative and a positive on the comparison card
  • a comparison card comprising at least a negative and a positive on the comparison card
  • the comparison card includes an example of at least 1, 2 or 3 in Figure 15A, and at least 1, 2 or 3 examples in Figure 15B; more preferably, the comparison card comprises Figure 15 All 6 examples.
  • determining whether the sample contains the misfolded protein or the misfolded protein aggregate comprises determining the result of the diffusion of the mixed solution on the microporous membrane by an automatic determination system to complete the detection result and outputting the judgment result.
  • the automatic determination system 200 includes a signal acquisition module 204 and a signal processing module 206.
  • the automatic determination system 200 may further include a user interaction module 202, wherein the modules are connected to each other by wire or wirelessly. Pass data or signals.
  • the signal acquisition module 204 includes an optical signal collector, such as a digital camera or scanner.
  • the user sends a signal acquisition command to the signal acquisition module through the user interaction module 202, and the signal acquisition module 204 obtains a picture signal of the diffusion condition by photographing or scanning the diffusion result, and then transmitting the collected image signal to the signal.
  • the processing module 204 compares the signal collected by the representative image with the comparison database, and obtains the determination result based on the specific algorithm and transmits the result to the user interaction module 202.
  • the comparison database contains a large amount of clinical sample detection data, and the signal processing module performs comparison by an intelligent algorithm.
  • signal processing module 204 delivers the determination to a third party system, such as a hospital's HIS or LIMS system.
  • the present invention provides an automatic detection system for detecting the presence or absence of misfolded proteins in a sample (e.g., maternal urine) or whether a pregnant woman has pre-eclampsia or has a risk of pre-eclampsia, including (1) an assembly of the present invention. , device or kit, and the above automatic determination system.
  • the automated detection system of the present invention comprises (1) an assembly, device or kit of the invention, and (2) the signal acquisition module comprises an optical signal collector, such as a digital camera or scanner, and 3)
  • the information memory is used to store the processed or unprocessed signals collected by the signal acquisition module, and the information memory may be a HIS system or a LIMS system.
  • An optical signal collector of the signal acquisition module such as a digital camera or scanner, transfers the acquired signal, ie, the diffusion result of the mixed solution on the microporous membrane (eg, in the form of photographs or digital information) to an HIS system or LIMS system or computer system.
  • the liquid outlet of the capillary tube and the above portion are filled with the above mixture, that is, a sample such as urine and a dye capable of competing with the microporous membrane and the misfolded protein to be mixed.
  • the volume of the mixed liquid is 1 to 30 ⁇ L, more preferably 1 to 25 ⁇ L, for example, about 1 to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 Or 16 ⁇ L; more preferably 8 to 15 ⁇ L.
  • the capillary since the capillary is in close contact with the microporous membrane and is in close contact, a certain amount of the mixed solution contained in the capillary is slowly released onto the microporous membrane, thereby causing micropores around the liquid outlet of the capillary.
  • the above-mentioned diffusion spots are formed on the surface of the film.
  • diffusion spots include those shown in Figure 15; in one embodiment, the dye collects from the capillary and the dye accumulates at the liquid outlet, does not diffuse and forms dark spots; in another embodiment, the mixing After the liquid is released from the capillary, the dye forms a dark spot near the liquid outlet, and there is a "pseudo-foot"-like diffusion; in another embodiment, the dye is released from the capillary and the dye is formed near the liquid outlet. Light color spread but contains pseudo feet. In another embodiment, after the mixture is released from the capillary, a small red spot is formed at the liquid outlet, but a distinct circular diffusion spot is formed around the mixture, or the spotted portion forms an outward diffusion of irregular diffusion. Spots, but no "pseudo-foot", or the formation of uniform, large circular diffusion spots.
  • the method of detecting whether a sample contains misfolded proteins or misfolded protein aggregates comprises the following steps:
  • a third capillary is used to suck a certain amount of the positive control solution, that is, the third mixture, and the liquid outlet of the third capillary is in close contact with the third microporous membrane, so that the third mixture in the third capillary is slowly released to the third On the microporous membrane.
  • the diffusion of the first, second, and third mixtures on the microporous membrane is observed to determine that 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 mixture of negative urine and dye.
  • the third mixed solution may be a dye solution determined to contain misfolded proteins, may be formed by mixing a positive sample determined to contain misfolded proteins with a dye, or by mixing a misfolded protein positive reference with a dye.
  • the above method can simultaneously detect a plurality of (2, 3, 4, 5, 10 or more) samples, that is, the plurality of samples are separately mixed with the dye to form a mixed solution, and then respectively formed by capillary spotting. The spots are diffused and the samples are judged to contain misfolded proteins based on the diffusion spots.
  • the assembly of the present invention may also include 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or more capillaries, each of which has a liquid outlet and a micropore The surface of the membrane is in contact with and close, and these capillaries can be held together by a connecting bridge to form a spotter composed of a row of capillary tubes.
  • the assembly 160A includes capillaries 162, 182, and 192 each having a liquid outlet 164, 184, and 194, respectively, and another open nozzle 166, 186, 196, an open tube.
  • the ports 166, 186, and 196 penetrate the connecting bridge so that both ends of the capillary are in communication with the outside.
  • the bridge 180 serves as a support and fixed capillary 162, 182, and 192, and the fluid outlets 164, 184, and 194 of the assembly 160A are in contact with and adhere to the surfaces of the microporous membranes 168, 188, 198, respectively.
  • the capillaries 162, 182, and 192 are each provided with a mixed solution.
  • the liquid in the capillary 162 is a mixture of the urine sample of the pregnant woman to be tested and the dye
  • the liquid in the capillary 182 is a negative control sample or buffer.
  • a mixture of liquid and dye, the liquid in capillary 192 is a mixture of a positive control sample and a dye.
  • Figure 16B shows that the liquid portion of the capillary of the assembly of Figure 16A above is absorbed by the microporous membrane;
  • Figure 16C shows that all of the liquid in the capillary of the assembly of Figure 16A above is absorbed by the microporous membrane, forming a different shape in the microporous membrane. Spot spread results.
  • Another aspect of the invention provides a device for detecting whether a sample contains misfolded proteins or misfolded protein aggregates.
  • a specific embodiment of the present invention may include a panel with two rows of grooves and a spotting member embedded in the panel and detachable from the panel.
  • the two rows of grooves on the panel there are one to more (3, 10, 12 or more) grooves in each row, one of which is a sample slot and the other row is a test slot.
  • the sample cell has a diameter of 0.3 to 1 cm, most preferably 0.5 cm, a depth of 0.3 to 1 cm, and most preferably 0.5 cm; the sample cell can hold 30-500 ⁇ L or more of liquid, preferably, more than 3 samples on the panel
  • the tank includes a sample tank to be tested, a negative control sample tank, and a positive control sample tank.
  • the sample tank to be tested may be filled with the above dye or dye solution or a mixture of the dye and the sample to be tested; the negative control sample tank may be filled with a negative control sample; the positive control sample tank may be provided with a positive control sample.
  • the sample tank can be sealed with a sealing material.
  • the test cell has a diameter of 0.5 to 2 cm, most preferably 1.8 cm, a depth of 0.2 to 1 cm, and most preferably 0.3 cm.
  • the bottom of the test tank is covered with the above microporous membrane such as filter paper.
  • the number of test slots on the panel is the same or more than the sample slot.
  • the spotting component embedded in the panel and detachable from the panel is as follows, specifically, one or more capillaries, which can be fixed on a beam or column, for example, 2 or more capillaries of the same length Parallel, side by side fixed to a beam, and the spacing between the capillaries corresponds to the spacing between the sample slots and the test slots so that the plurality of capillaries on the spotting component can be simultaneously inserted into the corresponding plurality of sample slots or correspondingly Test slots.
  • the detecting device of the present invention shown in FIG. 2 includes a test member including a microporous film, and the spotting member includes a capillary tube.
  • the number of the capillary tubes is two or more, for example, two or three. 4, 5, 6, 7, 8, 9, 10 or more.
  • the test component can be a separate microporous membrane.
  • the test component is comprised of a first cover and a first base, the first cover and the first base being either detachably or non-detachably coupled.
  • the first cover and the first bottom plate are non-detachably connected to be integrated, although the first cover and the first bottom plate are not physically distinguished, but for the convenience of description, distinguish.
  • the first cover is covered with a microporous membrane, as shown in Figures 4A, B.
  • the microporous membrane is one or more, for example, 2, 3, 4, 5, 10, 20, 50, 100, 200, and the microporous membrane may be of any shape. For example, a circle, an ellipse, a square, or an irregular shape.
  • one microporous membrane can only be diffused for one sample, and more preferably, the microporous membrane is circular with an area not less than the largest area that the sample can diffuse.
  • one microporous membrane can be diffused for 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, each of which can be diffused by one sample, and more preferably, the section is square, and the area of the inscribed circle is not less than the largest area that the sample can diffuse.
  • the first cover plate is provided with a test slot
  • the test slot is a cylindrical groove
  • the depth is 1/3 to 2/3 of the thickness of the sample-carrying member
  • the micro-hole is provided in the groove.
  • the membrane, the microporous membrane is circular, has a diameter slightly smaller than the cross-sectional diameter of the groove, and is laid flat at the bottom of the groove as shown in Figures 4C and D.
  • the number of test slots is plural, such as 2, 3, 5, 10, 15, 20, 30, 50, 100, 200, preferably Ground, one sample per cell for diffusion.
  • the first cover and the first bottom panel are detachably connected, optionally by providing a snap connection at the edges and/or corners, optionally by adhesive Connected, optionally, joined by soldering.
  • the first cover plate is provided with a circular hole, and a microporous film is disposed between the first cover plate and the first bottom plate, and the first cover plate and the first bottom plate are fastened or engaged or adhered After the combination, the circular hole on the first cover plate 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.
  • the detecting device further comprises a sample loading component consisting of a second cover plate and a second bottom plate, wherein the second cover plate is provided with a sample slot, and the sample slot is a cylindrical groove.
  • the lower surface of the second cover is detachably or non-detachably connected to the upper surface of the second base; preferably detachably connected, more preferably by locating at the edges and/or corners Connection, either by adhesive bonding or by soldering.
  • the side of the first cover is non-detachably connected to the side of the second cover as a whole, physically indistinguishable, and the side of the first bottom plate is non-detachably and the second bottom plate 22
  • the side connections are integrated into one and there is no physical difference.
  • a flow guiding groove is disposed between the first cover plate and the second cover plate, and the flow guiding groove is a long groove perpendicular to the center connection of the first cover plate and the second cover plate, and the groove is The shape of the cross section is inverted triangle or arc shape.
  • the purpose of the flow guiding groove is to prevent the liquid in the sample tank from flowing out and entering the test tank to produce deviation of the detection result.
  • the side of the first cover 11 is detachably coupled to the side of the second cover, or the side of the second base is detachably coupled to the side of the second base.
  • the side of the first cover is detachably coupled to the side of the second cover, and the side of the first base is detachably coupled to the side of the second base.
  • the joining means joining by means of the movable part 5, for example by a soft material or by a hinge, whereby the carrying part can be folded with the test part.
  • the shape of the sample loading member or the test member may be any shape, preferably one selected from the group consisting of a triangle, a quadrangle, a polygon, an ellipse, and a circle, and more preferably, a sample loading member or The shape of the test component is axisymmetric.
  • the number of sample slots is the same as the number of test slots
  • the sample tank and the test tank are arranged in a single row, in a plurality of rows, or in a polygonal arrangement, a circular arrangement, or an arbitrary arrangement, preferably in a straight line.
  • sample and test cells are arranged in the same manner.
  • the sample well and/or the test slot may be of any shape, preferably one selected from the group consisting of a triangle, a quadrangle, a polygon, an ellipse, and a circle.
  • the sample and test cells are circular.
  • the sample well has a diameter of from 0.3 to 1 cm, most preferably 0.5 cm, a depth of from 0.3 to 1 cm, and most preferably 0.5 cm.
  • the test cell has a diameter of from 0.5 to 2 cm, most preferably 1.8 cm, a depth of from 0.2 to 1 cm, and most preferably 0.3 cm.
  • the sample well and test slot are the same size.
  • the spotting member is a hollow box-like member containing a capillary tube.
  • the hollow box member is detachably or non-detachably coupled to the sample loading member and/or the test member, the hollow box member having an opening for facilitating access to the capillary, preferably the opening and the sample
  • the slot and the test slot opening are oriented the same, and more preferably, the opening is provided with a cover.
  • the spotting component is a solid box-like component having recessed holes therein for the placement of capillaries.
  • the solid box-like component is detachably or non-detachably coupled to the sample loading component and/or the test component.
  • the solid box-like component is integral with the sample-loading component and/or the test component.
  • a recessed hole is provided directly on the sample loading member and/or the test component for placing the capillary tube.
  • the recessed hole is one and can accommodate all the capillaries; preferably, the recessed hole can place a plurality of capillaries, more preferably Ground, the number of recessed holes is the same as the number of capillaries, and the diameter and depth of the recessed holes are slightly larger than the capillary tubes, and one capillary is placed in each recessed hole. The recess does not intersect the sample or test slot.
  • the spotting member is a spotter having a capillary on its side.
  • the body of the spotter is a rectangular parallelepiped, and the side of the rectangular parallelepiped is provided with a concave hole, and the concave hole penetrates the rectangular parallelepiped, so that both ends of the capillary tube communicate with the outside and the capillary tube is inserted into the concave hole.
  • the capillary and the rectangular parallelepiped are A whole.
  • the sample loading member and/or the test member 1 are provided with recessed holes into which the capillary projections of the spotting members can be inserted.
  • the number of capillaries is the same as the number of sample slots or the number of test slots, in the same manner as the sample well or test slot.
  • the capillary tubes are two or more, for example three, disposed on the same side of the cuboid body such that the spotting members assume a tandem capillary shape.
  • the side of the spotter setting capillary further comprises a positioning pin
  • the first cover plate is provided with a positioning hole
  • the second cover plate is provided with a positioning hole, when the positioning pin is inserted into the second cover plate
  • the capillary is inserted into the sample slot to suck the liquid.
  • the positioning pin is inserted into the positioning hole of the first cover
  • the capillary contacts the microporous membrane in the test slot, and the liquid in the capillary is adsorbed by the microporous membrane and is in the micropore Diffusion is carried out on the membrane.
  • the shape of the locating needle is selected from one of a bullet shape, a cylinder, a rectangular column, a triangular prism, an irregular column, any column shape, or any column shape that is inclined.
  • the locating pin is in the shape of a bullet.
  • the positioning needle is a hollow structure.
  • the positioning pins are positioned on either side of the spotter or in the middle of the capillary.
  • the number of locating pins is at least two, preferably two, three, four or more.
  • the number of positioning pins is two, which are respectively disposed on both sides of the spotter.
  • the outer surface of the sample loading component and/or the testing component is further provided with a concave hole arranged in the same manner as the capillary and the positioning pin on the spotter, so that the spotting can be performed
  • the device is fixed to the sample loading component or the testing component.
  • the spotter is provided with projections or grooves for the purpose of increasing friction and facilitating the handling, insertion or removal of the spotter.
  • one end of the capillary is coupled to a device that creates a negative pressure inside the capillary.
  • the means capable of generating a negative pressure inside the capillary is a rubber ball, like a dropper, in use, pinching the rubber ball while the other end of the capillary is placed in the liquid sample, releasing The rubber ball and the rubber ball are deformed to cause a negative pressure in the capillary, thereby allowing the liquid sample to enter the capillary more quickly.
  • the means capable of creating a negative pressure inside the capillary is a piston device such as a syringe, a pipette or the like.
  • the other end of the capillary is first placed in the liquid, and the piston is manually pulled or the piston is pulled by the elastic means to generate a negative pressure inside the capillary, so that the liquid sample enters the capillary more quickly.
  • the above-described means for generating a negative pressure inside the capillary can also cause a positive pressure inside the capillary to push the liquid in the capillary to slowly flow out, transfer to the microporous membrane, and complete the detection.
  • the sample loading component, the testing component, and the spotting component are made of PVC material, PUC material, nylon material, rubber material, acrylonitrile-butadiene-styrene copolymer material (ABS material), glass.
  • PVC material PUC material
  • nylon material nylon material
  • rubber material acrylonitrile-butadiene-styrene copolymer material (ABS material)
  • ABS material acrylonitrile-butadiene-styrene copolymer material
  • the metallic material is an alloy material, and more preferably, the alloy material is stainless steel.
  • the detecting device of the present invention has at least two core components, including a detecting member of the microporous film and a spotting member including a capillary. Therefore, those skilled in the art can understand the preparation method according to the disclosure of the specification: for the detecting component, the microporous film can be fixed on the supporting surface, and the three methods shown in FIG. 4 can be selected, and other methods can also be selected. method.
  • the spotting component if there are more than one capillary, for example three, the capillary can be connected by a connecting bridge, as shown in Fig. 1C, the capillary includes a liquid outlet and another open port, which penetrates the connecting bridge, so that another opening Also connected to the air.
  • the arrangement of the capillaries can be any other way.
  • Another aspect of the present invention provides a kit for detecting whether a sample contains a misfolded protein, including a microporous membrane and a capillary.
  • the kit comprises any of the above detection devices.
  • the detection device in the kit includes a panel with at least two rows of grooves and a spotting member embedded in the panel and detachable from the panel, wherein the two rows of grooves on the panel There are 1 to more (3, 10, 12 or more) grooves in each row, one of which is the sample slot and the other is the test slot.
  • there are more than 3 sample tanks on the panel including a sample tank to be tested (containing a mixture of dye or dye solution or dye and 30-500 ⁇ L or more of the sample to be tested) and a negative control sample tank ( A positive control sample well (containing at least 30 ⁇ L of positive control sample) was loaded with at least 30 ⁇ L of negative control sample.
  • the sample cell is sealed with a sealing material.
  • the bottom of the test tank is covered with the above microporous membrane such as filter paper.
  • the spotting component embedded in the panel and detachable from the panel comprises one or more capillaries, two or more capillaries of the same length being parallel, side by side fixed to a beam, and the spacing between the capillaries and the sample slots and The spacing between the test slots is such that a plurality of capillaries on the spotting member can be simultaneously inserted into the corresponding plurality of sample slots to pick up the liquid therein, and then simultaneously inserted into the corresponding plurality of test slots.
  • the kit comprises a dye capable of binding to a microporous membrane and misfolded proteins.
  • the dye is selected from one or more of a heterocyclic dye, Congo red, thioflavin, and Evans blue, preferably Congo red.
  • the dye is preloaded into the sample tank as a solid dry powder or solution.
  • the kit further comprises a control sample comprising a positive control sample and/or a negative control sample.
  • the control sample is preloaded into the control sample well.
  • the sample reservoir is sealed with a sealing material selected from the group consisting of tin foil, plastic film, and aluminum foil.
  • the seal can be glued or molded or any other method that can be sealed.
  • the negative control sample contains no misfolded proteins and the positive control sample contains misfolded proteins.
  • the positive control sample comprises any material having the same or similar structure as the misfolded protein, having a property capable of competing with the microporous membrane for binding to a dye (eg, Congo red), such as having a beta sheet structure.
  • Protein such as denatured bovine serum albumin (BSA).
  • the negative control sample is PBS buffer and the positive control sample is PBS buffer containing misfolded BSA.
  • the negative control sample is a urine sample that does not contain misfolded material and the positive control is a urine sample containing misfolded protein.
  • the kit may further comprise the above-described alignment card, desiccant, instructions.
  • the sample is from a patient's body fluid with a misfolded protein disease, including but not limited to whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid, chest and ascites, tears, vaginal secretions, semen , tissue lysate and combinations thereof.
  • a misfolded protein disease including but not limited to whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid, chest and ascites, tears, vaginal secretions, semen , tissue lysate and combinations thereof.
  • the misfolded protein disease is pre-eclampsia and the sample is maternal urine.
  • FIG. 1A This example provides the type of capillary used in the present invention.
  • the appearance of the capillary used in the present invention may be in a variety of shapes (Fig. 1A).
  • the cross-section of one end of the microporous membrane ie, one end of the liquid outlet
  • These capillaries can absorb 5-20 ⁇ L of liquid.
  • the speed of the liquid released onto the filter paper is not more than 4 ⁇ L/sec.
  • the capillary is connected by a connecting bridge 108, the liquid outlet 102 of the capillary 100 is used to suck the liquid and contact the filter paper, and the other open port 106 is connected to the air through the connecting bridge 108.
  • This embodiment provides a kit comprising the apparatus of Figures 2, 3, and 4, further comprising one or two stainless steel capillaries having an inner diameter of 1.25 mm.
  • the apparatus shown in FIG. 2 includes a test component 201 that includes filter paper 251. There are 3 ways to set the filter paper on the test part:
  • test component 201 is a rectangular parallelepiped, and the upper surface of the rectangular parallelepiped is covered with a circular filter paper 251 (the arrangement is as shown in FIG. 4A, B).
  • test component 201 is a rectangular parallelepiped, and the upper surface of the rectangular parallelepiped is provided with a cylindrical recess 231, and the bottom of the recess is provided with filter paper 251 (Fig. 4C, D).
  • the test component 201 includes a cover plate 211 and a bottom plate 212, and the cover plate is provided with a circular hole 271.
  • a layer of filter paper 251 is disposed in the middle (as shown in FIG. 4E, F).
  • the round holes form a test slot 231, and the bottom of the test slot is Filter paper 251.
  • the apparatus shown in FIG. 3 includes a test component 301 which is a rectangular parallelepiped having two test slots 311 and 312 on one side thereof.
  • the test slots 311 and 312 are each a cylindrical recess having a groove depth of about 2/3 of the thickness of the rectangular parallelepiped, the filter paper is provided in the groove, the filter paper is round, slightly smaller than the cross section of the groove, and is laid on the bottom of the groove (the arrangement is shown in Figure 4C, D, the filter paper is not shown in Figure 3) .
  • the specific detection procedure for detecting whether the pregnant woman's urine contains misfolded protein is that the sample taken from the pregnant woman's urine is mixed with Congo red to obtain a mixture, and the capillary is placed vertically in the mixed solution for about 10 seconds (10 ⁇ L can be taken up) The mixture is transferred to the test tank, and the outlet of the capillary is in close contact with the filter paper, and it is kept vertical, and stays for about 15 seconds. The liquid in the capillary is slowly released onto the filter paper, and the capillary is removed to observe the result.
  • the device of FIG. 3 can be used to detect two samples to be tested, or 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 was PBS buffer and the positive control sample was a PBS buffer containing a specially denatured treatment to form misfolded BSA.
  • This embodiment provides a kit and a method of using the same, and the kit includes the apparatus shown in FIG. 5, FIG. 6, FIG. 7, or FIG.
  • the detecting device shown in FIG. 5 includes a testing component 501 and a loading component 502.
  • the testing component 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 are test slots. 514, 516, 518, the bottom of the test slot is provided with filter papers 534, 536, 538 according to Figs. 4C, D.
  • the lower surface of the first cover 511 is non-detachably connected to the upper surface of the first bottom plate 512 to be integrated.
  • the sample loading component is composed of a second cover plate 521 and a second bottom plate 522, and the second cover plate is provided with three sample slots 525, 527, 529.
  • the lower surface of the second cover plate 521 is non-detachably connected to the upper surface of the second bottom plate 522 to be integrated; 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 of the 512 is directly connected to the side of the second bottom plate 522 to be integrated.
  • the test component and the sample loading component are integrated, and there is no physical difference.
  • the detecting device shown in FIG. 6 includes a testing component 601 and a loading component 602.
  • the testing component 601 is composed of a first cover 611 and a first bottom plate 612.
  • the upper surface of the first cover 611 is provided with five test slots 631 and 633. 635, 637, and 639, filter papers 651, 653, 655, 657, and 659 are respectively arranged at the bottom of the test slot (the arrangement is as shown in FIG. 4C and D).
  • the sample loading component is composed of a second cover plate 621 and a second bottom plate 622, and the upper surface of the second cover plate is provided with five sample slots 641, 643, 645, 647, 649.
  • the side surface of the first cover plate 611 is connected to the side surface of the second cover plate 621 by a 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. Thereby, the test component 601 and the sample loading component 602 can be folded together during transportation and storage, thereby saving space.
  • the detecting device shown in FIG. 7 includes a testing component 701 and a loading component 702.
  • the upper surface of the testing component is provided with three test slots 731, 733 and 735, and the bottom of the test slot is respectively provided with filter papers 751, 752 and 753 (setting manners such as Figure 4C, D)).
  • the upper surface of the sample loading member 702 is provided with three sample grooves 741, 743, and 745.
  • Test component 701 and load bearing component 702 are coupled by a living hinge 705.
  • the detection apparatus shown in Fig. 8 is substantially the same as that of Fig. 7, except that the number of test slots and sample slots is larger (14) than that shown in Fig. 7, so that it can be used to detect more samples.
  • Either kit also included a stainless steel capillary having the same number of sample slots and an inner diameter of 1.25 mm.
  • the specific detection step for detecting whether the pregnant woman's urine contains misfolded protein is that the sample taken from the urine of the pregnant woman and the Congo red (concentration 5 mg/mL) are mixed in the sample tank at a ratio of 20:1 to obtain a mixed solution. Place it vertically in the mixture for about 10 seconds (can draw about 10 ⁇ L of the mixture), transfer the capillary to the test tank, make the liquid outlet contact with the filter paper, stay for about 15 seconds, and the liquid in the capillary is slowly released onto the filter paper. Remove the capillary and observe the result.
  • up to three samples to be tested can be detected. Of course, one sample to be tested can also be detected, and the other two test slots are used to detect the negative control sample and the positive control sample.
  • the detection method is the same as this.
  • the device of FIG. 6 can be used to detect up to 5 samples to be tested, or to detect 3 samples to be tested, 2 control samples, ie, a negative control sample and a positive control sample.
  • up to 14 samples to be tested can be detected, or 12 samples to be tested, 1 negative control sample, and 1 positive control sample can be detected.
  • This embodiment provides a kit and a method of using the same, and the kit includes the apparatus shown in FIG. 9, FIG. 10 or FIG.
  • the detecting device shown in FIG. 9 includes a test member 901, a sample loading member 902, and a spotting member 903.
  • the test component is composed of a first cover plate 911 and a first bottom plate 912 (not shown), and the first cover plate 911 is provided with 14 test slots 931.
  • the sample loading member 902 is composed of a second cover plate 921 and a second bottom plate 922 (not shown), and the second cover plate 921 is provided with 14 sample slots 941.
  • the first cover plate 911 and the first bottom plate 912 are integrally formed and physically indistinguishable, and the filter paper 951 is disposed in the test slot 931, and is disposed in the manner shown in FIG. 4C and D.
  • the second cover plate 921 and the second bottom plate 922 are integral and physically indistinguishable.
  • the test component 901 and the sample loading component 902 are connected together by a snap 905, and can be stacked separately when transported and stored, and connected together when in use.
  • the spotting member 903 is a hollow box-like member including a capillary 933 which is a glass tube having an inner diameter of 1.2 mm, and the hollow box-shaped member is connected to the sample loading member 902 and the test member 901 by a snap connection.
  • the detecting device shown in FIG. 10 includes a test component 1001, a sample loading component 1002, and a spotting component 1003.
  • the test component is composed of a first cover 1011 and a first bottom plate 1012.
  • the first cover 1011 is provided with five test slots 1031.
  • the sample loading component is composed of a second cover plate 1021 and a second bottom plate 1022, and the second cover plate 1021 is provided with five sample slots 1041.
  • the first cover 1011 and the first bottom plate 1012 are integrally formed and physically indistinguishable, and the filter paper 1051 is disposed in the test slot 1031 in the manner of FIG. 4C and D.
  • the second cover plate 1021 and the second bottom plate 1022 are integral and physically indistinguishable.
  • the spotting member 1003 is a solid box-shaped member on which as many recessed holes 1032 as the number of sample slots are provided, the diameter of the recessed holes 1032 being slightly larger than the diameter of the capillary (not shown), and the depth is slightly larger than the length of the capillary.
  • One capillary can be placed in each recess.
  • the upper surface of the solid box-shaped member is non-detachably connected to the lower surface of the first bottom plate 1012 and the lower surface of the second bottom plate 1022, and is integrated.
  • the detecting device shown in FIG. 11 includes a test component 1101, a sample loading component 1102, and a spotting component 1103.
  • the test component 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, and the second cover plate 1121 There are five sample slots 1141.
  • the detecting device is a whole, but the division is made to distinguish, and the first cover 1111 is provided with a test slot 1131, and the filter paper is also placed in the same manner as in Figs. 4C and D.
  • the spotting member 1103 is provided with a recessed hole 1151 for accommodating a capillary 1153 having a number of sample slots (a stainless steel capillary having an inner diameter of 1.25 mm) having a depth slightly larger than the length (3 cm) of the capillary 1153.
  • This embodiment provides a kit and a method of using the same, which includes the apparatus shown in FIGS. 12, 13, and 14.
  • the detecting device shown in Fig. 12 includes a test member 1201, a sample loading member 1202, and a spotting member 1203, all of which are made of a PVC material.
  • the test component is composed of a first cover 1211 and a first bottom plate 1212 (not shown).
  • the sample loading component is composed of a second cover plate 1221 and a second bottom plate 1222 (not shown), and the second cover plate 1221 is provided with three sample slots 1241.
  • the side of the first cover 1211 is non-detachably connected to the side of the second cover 1221 to be integrated, and a guide groove 1206 is disposed between the first cover 1211 and the second cover 1221, and the guide groove 1206 and the test component
  • the 1201 and the carrier member 1202 are perpendicular to the center line and have an inverted triangular shape in cross section, extending through the entire upper surface.
  • the sides of the first bottom plate 1212 are non-detachably connected to the sides of the second bottom plate 1222 to be integrated.
  • the first cover 1211 is provided with three circular holes. When connected with the first bottom plate 1212, a layer of filter paper is placed in the middle (as shown in FIG. 4E, F), so that the first cover 1211 and the first bottom plate 1212 pass the four-corner card. After the buckle is closed, the round hole forms a test slot 1231, and the bottom of the test slot is the filter paper 1251.
  • the spotting part 1203 is a spotting plate, and the side of the spotting plate is provided with a capillary 1233.
  • the number of the capillary is the same as the number of sample slots or the number of test slots, that is, three, and the arrangement is the same as that of the sample slot or the test slot;
  • the same side of the capillary tube, the two sides of the capillary tube further include a positioning pin 1235, and the first cover plate 1211 and the second cover plate 1221 are provided with positioning holes 1204 and 1208.
  • the positioning pin 1235 is inserted into the positioning hole 1204 or 1208, the capillary 1233 It can be inserted into the sample slot 1241 or the test slot 1231.
  • the loading member 1202 is further provided with a concave hole (not shown) with respect to the side surface of the testing member 1201, and the concave hole is arranged in the same manner as the capillary 1233 and the positioning pin 1235 on the spotting plate, so that the positioning member 1203 can be fixed at the same.
  • a protrusion 1237 is also arranged on the sample plate to facilitate insertion and removal of the sample plate.
  • the detecting device shown in Fig. 13 is basically the same as the detecting device shown in Fig. 12, except that the number of test slots and sample slots is larger than that shown in Fig. 12 (5), which can be used to detect more samples. .
  • the kit is used as follows:
  • the spotted part shows a more concentrated, non-diffusing red spot (Fig. 15A, a), or a pseudo-foot, in addition to the spotted part showing a more concentrated, non-diffusing red spot. (Fig. 15A, b), or with a slight spread (Fig. 15A, c), the maternal urine sample was judged to be negative, i.e., did not contain misfolded proteins or contained misfolded proteins below the reference value.
  • the spotted portion shows a small red spot, but there is a relatively obvious diffusion around (Fig. 15B, d), or the spotted portion shows a red spot, and a shallowly colored circle with irregular diffusion ( Figure 15B, e), or the formation of a uniform, large diffusion spot ( Figure 15B, f), the pregnant woman urine sample is judged to be positive, that is, contains misfolded protein or contains misfolded protein higher than the reference value.
  • the detecting device provided by the embodiment integrates the equipment and the reagent, greatly compresses the packaging space, is convenient for transportation, storage, and is convenient for laboratory operation.
  • This embodiment provides a simple kit and a method of using the filter paper and capillary tube (stainless steel capillary having an inner diameter of 1.25 mm).
  • the method of use is as follows:
  • Fig. 16 is a view showing the detection process of the detecting device of the present invention. After the capillary contacts the filter paper, the sample is slowly released on the filter paper and spread outward at the contact position to form dye spots of different sizes.
  • the capillary used was a glass capillary with a circular outlet having an inner diameter of 1.25 mm and a length of 10 cm, and the capillary was marked with a 10 ⁇ L mark.
  • the pipette used was a 10 ⁇ L pipette.
  • the dropper used was 100 ⁇ L, and 10 ⁇ L of liquid was taken in each test.
  • the negative sample is the urine sample of the pregnant woman who is judged to be non-eclamptic according to the clinical result.
  • the positive sample is the urine sample of the pregnant woman who is judged as pre-eclampsia according to the clinical result.
  • Congo red was added in a ratio of 50:1 in the negative and positive samples, respectively. Insert the capillary into the negative or positive sample respectively. When the liquid rises to the 10 ⁇ L mark, take out the capillary and touch the liquid to the filter paper. After the sample is absorbed by the filter paper, remove the capillary and spread for about 15 seconds. result.
  • the capillary, the pipette and the dropper are used to draw 10 ⁇ L of the negative sample or the positive sample, respectively, and the capillary outlet, the pipette tip or the dropper tip. Leave it at 0.5 ⁇ 2cm above the filter paper, and release the extracted mixture onto the filter paper. After a period of diffusion, observe the test results.
  • the diffusion spot radius of the negative sample and the positive sample were measured separately, and the diffusion spot radius was the length from the center point of the spot to the point where the spot spread to the farthest position. And calculate the radius ratio of the diffusion radius of the positive sample to the diffusion radius of the negative sample. Each experiment was repeated 5 times.
  • the positive sample will form a uniform, large diffusion spot with almost no difference.
  • the negative samples formed relatively large spots, and the same spotting method as the positive samples formed the radius of the spots to be about 1.2. Smaller, it is easy to form a false positive.
  • the spots formed by the capillary detection are more concentrated, do not spread outward or rarely diffuse, and the ratio of the spot radius formed with the positive sample is 2.5 or more.
  • the pipette and the dropper are in contact with each other.
  • the diffuse spots formed by the negative sample are relatively large and have irregular outward spread, and the radius ratio is about 1.6.
  • capillary contact spot detection has the best effect, the distinction is more obvious, and it is not easy to form false positives, and the results are intuitive and reliable.
  • the capillary used was a stainless steel capillary having an inner diameter 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, and the capillary was marked with a 10 ⁇ L mark.
  • the negative sample is the urine sample of the pregnant woman who is judged to be non-eclamptic according to the clinical result.
  • the positive sample is the urine sample of the pregnant woman who is judged as pre-eclampsia according to the clinical result.
  • Congo red was added to the negative and positive samples, respectively. Insert the capillaries with different inner diameters into the negative or positive samples, and stay for more than 30 seconds. When the liquid no longer rises, take out the capillary and touch one end of the liquid to the filter paper until the sample is absorbed by the filter paper (about 3-10 seconds). , remove the capillary, diffuse for a period of time, observe the test results.
  • the diffusion spot radius of the negative sample and the positive sample were measured separately, and the diffusion spot radius was the length from the center point of the spot to the point where the spot spread to the farthest position. And calculate the radius ratio of the diffusion radius of the positive sample to the diffusion radius of the negative sample. Each experiment was repeated 5 times.
  • the spot diffusion of spots with different inner diameter capillaries is shown in Fig. 18 or Table 2. It can be seen that when the inner diameter of the capillary is too small (for example, less than 0.7 mm), the diffusion of liquid from the capillary to the filter paper is too slow, and the spot diffusion of the positive sample The radius is too small and does not differ much from the spot diffusion radius of the negative sample. As the inner diameter of the capillary increases, the increase in spot spread of the negative sample is not significant, but the spot spread of the positive sample increases rapidly.
  • the inner diameter of the capillary increases to a certain extent (for example, greater than 3 mm), since the contact area of the liquid with 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 not being Then increase or even decline.
  • the capillary is further enlarged, the liquid can no longer enter the capillary autonomously, resulting in failure to detect.
  • the capillary inner diameter is 0.7-2.8mm, the detection effect is better, and when the thickness is 1.12-2.64mm, the detection effect is better.
  • the capillary used was a glass capillary having an inner diameter of 1.25 mm and a length of 5 cm.
  • the maximum capillary volume of the capillary was 20 ⁇ L, and the capillary was marked with a scale of 1-20 ⁇ L and an accuracy of 1 ⁇ L.
  • the negative sample is the urine sample of the pregnant woman who is judged to be non-eclamptic according to the clinical result.
  • the positive sample is the urine sample of the pregnant woman who is judged as pre-eclampsia according to the clinical result.
  • Congo red was added to the negative and positive samples, respectively. Insert the capillary into the negative sample or the positive sample, respectively, and take 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 ⁇ L of liquid, and then take out the capillary. One end of the suction liquid is contacted with the filter paper, and the sample is all absorbed by the filter paper, the capillary is removed, and after diffusion for a while, the detection result is observed.
  • the diffusion spot radius of the negative sample and the positive sample were measured separately, and the diffusion spot radius was the length from the center point of the spot to the point where the spot spread to the farthest position. And calculate the radius ratio of the diffusion radius of the positive sample to the diffusion radius of the negative sample. Each experiment was repeated 5 times.
  • the capillary adsorption time becomes longer and longer, and since the negative sample is not diffused or diffused to a small extent, the liquid in the capillary is difficult to be completely adsorbed onto the filter paper or takes longer. It is completely adsorbed, causing the detection time to be too long.
  • the amount of spotting is at least 4 ⁇ L, and the effect is better at 5-15 ⁇ L.

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Abstract

一种检测样本中是否含有错误折叠蛋白或错误折叠蛋白聚集物的装置以及试剂盒和方法，其中检测装置包括测试部件和点样部件，测试部件包含微孔膜，点样部件包含毛细管。试剂盒包括能够与错误折叠蛋白和微孔膜结合的染料；检测时，将待测样本与染料混合，形成混合液，利用毛细管吸取该混合液，后将毛细管出液口与微孔膜充分接触，混合液从毛细管中缓慢释放到微孔膜上并将微孔膜染色，通过观察颜色的扩散情况判断待测样本中是否含有错误折叠蛋白或错误折叠蛋白聚集物。检测结果可通过肉眼或仪器识别，用于检测孕妇样本以预测、筛查、诊断孕妇子痫前期和子痫。

Description

一种检测错误折叠蛋白质的装置、试剂盒和方法 技术领域
本发明涉及一种检测装置和检测方法，具体地，涉及一种检测生物样本中错误折叠蛋白的装置和方法。
背景技术
蛋白质精确无误的天然折叠是一个复杂且容易出错的过程，即容易出现错误折叠，错误折叠蛋白就是由于错误折叠而形成的蛋白质，其与天然折叠形成的蛋白质构象不同。
蛋白错误折叠形成大量的排列成反向的β-折叠片层(β-sheet)结构。该结构由于疏水性表面暴露，导致蛋白质分子之间产生高度的粘着性，与其它错误折叠蛋白的β-Sheet结构聚集起来，聚集在一起的蛋白质分子，作为结晶核，使其他不相关的蛋白质共聚合形成错误折叠蛋白的聚集物，可容纳几乎无限的多肽链(Misfolded Protein Aggregates:Mechanisms,Structures and Potential for Disease Transmission，Semin Cell Dev Biol.2011July；22(5):482–487.)，形成寡聚体和纤维状聚集物。
这些聚集物已经发现与多种老年人相关的神经退行性疾病相关，像阿尔兹海默症、帕金森等都是错误折叠蛋白造成的，虽然这些蛋白都不一样(阿尔兹海默症的amyloidβ肽、帕金森病的α-Synuclein蛋白、亨廷顿舞蹈症的Huntingtin蛋白、疯牛病的Prion蛋白)，它们错误折叠后形成β片层(β-sheet)结构，都能形成上述聚集物，具有相同的构象。
而最近的研究发现子痫前期患者的尿液和胎盘组织中含有大量错误折叠蛋白及其聚集物，而正常孕妇中则没有这种聚集物，从而发现子痫前期和子痫的发病和这些错误折叠蛋白及其聚集物相关。(Buhimischi et al.,Science Translational Medicine,2014,6(245):245ra92)。然而，目前临床上还没有利用这 种标志物检测子痫前期的快速POC检测试剂盒。
发明内容
为了解决上述技术问题的至少一个，本发明的第一方面提供了一种检测样本中是否含有错误折叠蛋白质的装置，包括测试部件和点样部件，测试部件包含微孔膜，点样部件包含1、2、3个或更多个毛细管。进一步地，测试部件包括第一盖板和第一底板，所述第一盖板包含微孔膜。
进一步地，该装置还包括载样部件，其上设有一个或多个样本槽用于盛装样本(检测样本、阴性对照样本、和/或阳性对照样本)和/或染料。
在本发明的实施方案中，第一盖板上覆盖微孔膜。
在本发明的实施方案中，第一盖板上设置测试槽，测试槽中含有微孔膜。
根据本发明，任选地，点样部件为一空心盒状部件，其中含有1、2、3个或更多个毛细管；任选地，点样部件为一实心盒状部件，其上设有1、2、3个或更多个凹孔，用于放置相应的1、2、3个或更多个毛细管；任选地，点样部件为点样板，包括主体部分和毛细管，主体部分为板状部件，毛细管设置在侧面，毛细管数目为1、2、3个或更多个。毛细管一端为取液端，用于吸取和释放液体，另一端与点样板主体相连；任选地，点样部件为毛细管。
本发明的第二方面提供一种检测样本中是否含有错误折叠蛋白质的试剂盒，包括本发明第一方面所述的装置，还包括能够结合微孔膜和错误折叠蛋白的染料，优选地，染料选自杂环染料、刚果红、硫磺素、伊文思蓝中的一种或多种，更优选地，染料为刚果红。
本发明第三方面提供一种检测样本中是否含有错误折叠蛋白的试剂盒，包括微孔膜和1、2、3个或更多个毛细管。在本发明的实施方案中，还包括能够结合微孔膜和错误折叠蛋白的染料，优选地，染料选自杂环染料、刚果红、硫磺素、伊文思蓝中的一种或多种，更优选地，染料为刚果红。
进一步地，上述试剂盒还包括对照样本，对照样本包括阴性对照样本和/或阳性对照样本。
本发明的第四方面提供一种检测样本中是否含有错误折叠蛋白的方法，包 括以下步骤：
(1)将样本和能够与微孔膜和错误折叠蛋白结合的染料进行混合形成混合液，
(2)利用毛细管吸取至少5μL的混合液；
(3)将毛细管的出液口与微孔膜紧密接触，使得毛细管中的混合液被微孔膜吸出而在微孔膜里缓慢扩散，从而缓慢地完全释放到微孔膜里；
(4)基于染料的颜色，观察混合液在所述微孔膜上的扩散情况确定样本中是否含有错误折叠蛋白。
优选地，混合液是通过毛细管的虹吸而无外力作用进入毛细管。优选地5～25μL，更优选地8～15μL混合液吸入毛细管并完全释放到微孔膜里。
本发明的第五方面提供一种检测样本中是否含有错误折叠蛋白质的组合体，包括毛细管和微孔膜，其中，毛细管的出液口与微孔膜的表面接触并密合。
进一步地，毛细管中包含一定量能够结合微孔膜和错误折叠蛋白的染料和样本混合而成的混合液，所述毛细管的出液口与微孔膜紧密接触并密合，优选地，所述染料选自杂环染料、刚果红、硫磺素、伊文思蓝中的一种或多种，更优选为刚果红，一定量指5μL以上，优选地，为5～25μL，更优选地8-15μL。
进一步地，在所述微孔膜的表面、所述毛细管的出液口的周围由于毛细管内一定量的刚果红和样本的混合液自所述毛细管出液口缓慢释放而染色。
附图说明
图1A显示了本发明中使用的部分毛细管类型(A：毛细管外形，从左到右依次为：上下等粗毛细管，上粗下细毛细管，分段等粗毛细管，分段上粗下细毛细管，波浪形毛细管，五棱柱型毛细管，五角星型毛细管)，图1B显示了毛细管与微孔膜接触一端横截面可选择的其他形状，图1C显示了3个毛细管制成联排毛细管的示意图，上图左是主视图，上图右是俯视图，下图是左视图。
图2显示了本发明的测试部件一个具体结构的示意图。
图3显示了本发明的测试部件又一个具体结构的示意图。
图4显示了微孔膜4的设置方式，A、C、E分别是设置示意图，B、D、F分 别是相应的剖面图。
图5显示了本发明的检测装置一个具体结构的示意图(A：俯视图，B：正视图，C：左视图)。
图6显示了本发明的检测装置另一个具体结构的示意图(A：俯视图，B：正视图，C：展开时的左视图，D：折叠时的左视图)。
图7显示了本发明的检测装置另一个具体结构的示意图(A：俯视图，B：仰视图)。
图8显示了本发明的检测装置另一个具体结构的示意图。
图9显示了本发明的检测装置另一个具体结构的示意图。
图10显示了本发明的检测装置另一个具体结构的示意图(A：俯视图，B：正视图，C：左视图)。
图11显示了本发明的检测装置另一个具体结构的示意图(A：俯视图，B：正视图，C：左视图)。
图12显示了本发明的检测装置另一个具体结构的示意图。
图13显示了本发明的检测装置另一个具体结构的示意图。
图14显示了本发明的检测装置一个具体结构的使用方法示意图。
图15显示了本发明的检测装置或试剂盒检测结果。
图16示出了本发明利用联排毛细管进行检测的示意图。
图17显示了利用毛细管、移液枪和滴管点样对斑点扩散的影响。
图18显示了不同毛细管内径对斑点扩散的影响。
图19显示了不同点样量对斑点扩散的影响。
图20显示了本发明自动判定系统示意图。
发明详述
术语的定义
本发明所述的“错误折叠蛋白”或“错误折叠蛋白质”是相对于正确折叠的蛋白质而言。蛋白质错误折叠形成大量的排列成反向的β-折叠片层(β-sheet)结构,这些β-Sheet结构由于疏水性表面暴露，导致蛋白质分子之间产生高度的粘 着性，与其它错误折叠蛋白的β-Sheet结构自然就聚集起来，聚集在一起的蛋白质分子，作为结晶核，使其它不相关的错误折叠蛋白质共聚合形成错误折叠蛋白质的聚集物(寡聚体和纤维状聚集物)。在本发明中，“错误折叠蛋白质”的含义包括错误折叠蛋白形成的这些聚集物。
例如，研究发现，子痫前期患者的尿液和胎盘组织中含有大量错误折叠蛋白及其聚集物，能够与刚果红特异性结合，在电镜下观察发现这些嗜刚果红性的错误折叠蛋白聚集物具有非常类似于淀粉样蛋白(amyloid-like)的纤维状结构，而正常孕妇中则没有这种聚集物(Buhimschi et al.,SCIENCE Translational Medicine,2014,6(245):245ra92)。
不同的蛋白质即使在结构上和序列上并无同源性，一旦发生构象变化而错误折叠却不能修复或排除，都可形成β片层(β-sheet)结构，不同蛋白来源的β片层结构能相互作用形成包含不同蛋白的错误折叠蛋白聚集物，能构象特异性地结合刚果红。
本发明所述的“微孔膜”是指一类微孔材料制成或者表面覆盖微孔材料的薄膜，所述微孔材料因其空间结构和/或组成特殊性，而具有这样的功能：能够与错误折叠蛋白质竞争结合染料，即如果样本中不含错误折叠蛋白质，染料与微孔材料结合，从而使得染料不能或很难随溶剂一起在微孔膜上进行扩散，形成较小的有色斑点；如果染料与错误折叠蛋白质结合，则不与微孔材料结合，因此染料与错误折叠蛋白质结合产物能够在微孔膜上进行扩散，从而形成较大的有色斑点。微孔材料可以是任何本领域技术人员所熟知包含大量自由羟基的材料，例如纤维素，则微孔膜是纤维素膜，例如滤纸。染料可以是任何本领域技术人员所熟知的具有以上特性的，能够与错误折叠蛋白质构象特异性地结合，例如刚果红。
本发明所述的“毛细管”是指出液口内径不超过3.5mm或出液口横截面的面积不超过9mm 2的中空管，当毛细管插入到液体中时，液体能够浸润毛细管内表面，表现为毛细管中液面高度高于外部液面高度，即产生“毛细现象”，当毛细管离开液体时，由于表面张力而留在毛细管内部的液体不少于5μL，优选地，毛细管内表面具有亲水性。
毛细管的材料和质地或处理可以是任何相关领域内的技术人员所了解的能够达到上述浸润液体体积的毛细管。
在上述微孔膜上基于毛细现象的斑点扩散方法检测错误折叠蛋白质的过程中，利用能够与错误折叠蛋白质构象特异性地结合的染料例如刚果红溶液在微孔膜例如滤纸上扩散面积的大小，来检测是否存在错误折叠蛋白质。
但是，发明人发现，如果用移液器(如吸管、移液枪等)进行滴样，液滴直接滴落在滤纸上，与滤纸形成较大的接触面并且迅速在滤纸上扩散，这样即使不含错误折叠蛋白质的阴性样本在滤纸上也显现出较大的显色斑点，往往与阳性样本的显色斑点区分不大，导致检测结果不准确，造成很多假阳性和假阴性。即使所用吸管、移液管的出液口很小也无法克服这个缺陷。发明人进一步地发现，如果采用毛细管点样(而不是滴样)，用毛细管吸取足够体积的染料和样本的混合液，使毛细管的出液口与滤纸紧密接触，使毛细管中的混合液因滤纸材料的吸水性缓慢地释放到滤纸上，可以显著增加阴阳性样本产生的显色斑点的差异，大大减少假阳性率，显著提高检测准确率。
检测方法和组合体
本发明提供一种检测样本中是否含有错误折叠蛋白质或错误折叠蛋白质聚集物的方法，包括以下步骤：
(1)将样本和能够与微孔膜和错误折叠蛋白竞争结合的染料进行混合形成混合液，
(2)用毛细管吸取一定量的混合液；
(3)将毛细管的出液口与微孔膜紧密接触从而使毛细管中的混合液缓慢释放到微孔膜上；
(4)基于染料的颜色，观察混合液在所述微孔膜上的扩散情况以判定样本中含有错误折叠蛋白质或错误折叠蛋白质聚集物，优选地，当扩散面积超过参考值，判定样本中含有错误折叠蛋白质或错误折叠蛋白质聚集物。
另一方面，本发明提供一种检测样本中是否含有错误折叠蛋白聚集物的组合体或装置，可以用于实施上述检测方法。一般来说，组合体包括毛细管和微 孔膜，其中，所述毛细管的出液口与所述微孔膜的表面接触并密合。
具体地说，本发明的方法和组合体可以用于检测多种样本，例如全血、血清、血浆、尿液、唾液、汗液、脑脊液、胸腹水、泪液、阴道分泌物、精液、组织裂解液及其组合，当样本含有血液而呈颜色，可以在检测前进行离心去除红细胞或其它干扰因素。特别地，本发明的方法和组合体尤其适用于检测孕妇尿液中是否含有错误折叠蛋白质或错误折叠蛋白质聚集物，从而预测、检测、筛查或诊断孕妇是否患有子痫前期或有子痫前期风险。
优选地，在本发明中的微孔膜可以是由含有自由羟基的材料制成的薄膜并具有吸水性。在优选的实施方案中，微孔膜是由含有自由羟基的纤维素制成的纤维素膜，例如滤纸、书写纸、打印纸、标签纸。
染料可以是任何能够构象特异性地结合错误折叠蛋白质的染料，同时这种染料应该还能竞争性地结合微孔膜中的材料(例如含自由羟基的纤维素)。在优选的实施方案中，染料是刚果红。在其它实施方案中，染料是硫磺素或伊文思蓝。染料可以是固体染料直接与样本例如尿液混合并溶于液体样本，也可以是以溶液的形态与样本例如尿液混合，形成的混合液中染料例如刚果红的浓度可以是0.01～2mg/mL，优选地为0.02～1mg/mL，更优选地为0.05～0.5mg/mL。
在本发明中，毛细管沿着管长方向具有两端，一端为出液口，另一端可以是开口的也可以是封闭的，毛细管的出液口内径小于或等于3.5mm或者出液口的横截面的面积不超过9mm 2。毛细管应该有足够的管长而能够吸取和容纳至少4μL混合液，优选地至少5μL混合液、至少8μL混合液，更优选地8到15μL左右。在本发明的具体实施方案中，毛细管摄取的一定量混合液为2～30μL，4～30μL，更优选，为5-25μL、5-20μL、4-17μL、或者5-17μL，例如为5、6、7、8、9、10、11、12、13、14、15或16μL；更优选地8～16μL。优选地，毛细管沿着管长方向粗细大体一致，横截面包括出液口横截面可以是圆形或其它任何规则或不规则形状，例如那些在图1显示的。在优选实施方案中，出液口内径大约为0.5～3mm，0.7到3mm，优选地0.9到2.8mm，或横截面的面积大约为0.2～7mm 2，优选地约为0.64到6.2mm 2。在优选实施方案中，毛细管吸取混合液是通过毛细现象，吸取至少5μL液体。在优选实施方案中，毛细管吸取混合液后，将出液 口与微孔膜接触时，液体靠微孔膜的吸水性被动从毛细管释放到微孔膜里。优选地，液体释放到微孔膜中的速度不大于4.5μL/秒，不高于4μL/秒，优选地为0.5～4μL/秒，例如为0.5、1、1.5、2、2.5、3、3.5或4μL/秒，更优选为1～3μL/秒，例如1.2、1.6、1.7、1.8、2.3、2.6或2.8μL/秒。
当样本中不含有错误折叠蛋白质或错误折叠蛋白质聚集物，利用本发明的方法、组合体、装置或试剂盒得到的结果为阴性，即利用毛细管将样本与染料形成的混合液点样到微孔膜上后，在微孔膜上不形成明显的扩散，或者在一些实施方案中形成较小的扩散斑点而扩散半径小于参考值，该参考值可以由一定数量的(例如50-100个)临床判定为阴性的病人的样本形成的扩散斑点的半径的最大值来确定。
当样本中含有错误折叠蛋白质或错误折叠蛋白质聚集物，利用本发明的方法、组合体、装置或试剂盒得到的结果为阳性，即利用毛细管将样本与染料形成的混合液转移至微孔膜上后，在微孔膜上形成大于阴性样本的扩散斑点。在本发明的一些实施方案中，阳性样本产生较大的扩散斑点，扩散半径大于或等于一个特定的参考值，而该参考值可以由一定数量的(例如50-100个)阳性样本形成的扩散斑点的半径的最小值来确定。值得说明的是，在一些样本测试中，会产生类似“伪足”状的扩散，即使扩散斑点的半径大于阴性样本的扩散半径，或大于上述参考值，这种类型的扩散仍然判断为阴性。
在一个优选实施方案中，判定样本中是否含有错误折叠蛋白质或错误折叠蛋白质聚集物包括将混合液在上述微孔膜上的扩散结果与一比对卡比较，比对卡上包括至少阴性和阳性样本扩散结果的示例；优选地，比对卡包括图15A中至少1、2或3个的示例，以及图15B中至少1、2或3个示例；更优选地，比对卡包括图15中所有6个示例。
在一个优选实施方案中，判定样本中是否含有错误折叠蛋白质或错误折叠蛋白质聚集物包括将混合液在所述微孔膜上的扩散结果通过自动判定系统完成检测结果判断并输出判断结果。例如，如图20所示，自动判定系统200包括信号采集模块204和信号处理模块206，优选地，自动判定系统200还可以包括用户交互模块202，其中，模块之间通过有线或无线相互连接并传递数据或信 号。信号采集模块204包括光信号采集器，例如数码相机或扫描仪。使用时，用户通过用户交互模块202向信号采集模块发送信号采集命令，信号采集模块204通过对上述扩散结果进行拍照或扫描，获得扩散情况的图片信号，之后，将采集到的图像信号传输到信号处理模块204，信号处理模块将收集到代表图像的信号与对比数据库进行比对，基于特定的算法，得到判定结果并传输到用户交互模块202。优选地，对比数据库包含大量的临床样本检测数据，信号处理模块通过智能算法进行对比。任选地，信号处理模块204将判定结果输送到第三方系统中，例如医院的HIS或LIMS系统。
因此，本发明提供了一个用于检测样本(例如孕妇尿液)中是否存在错误折叠蛋白质或者孕妇是否患有子痫前期或者有子痫前期风险的自动检测系统包括(1)本发明的组合体、装置或试剂盒，以及上述自动判定系统。在另一具体实施例中，本发明的自动检测系统包括(1)本发明的组合体、装置或试剂盒，(2)信号采集模块包括光信号采集器，例如数码相机或扫描仪，以及(3)信息存储器用于储存用信号采集模块采集到的经过处理或未经处理过的信号，信息存储器可以是HIS系统或LIMS系统。信号采集模块的光信号采集器，例如数码相机或扫描仪，将采集的信号即混合液在所述微孔膜上的扩散结果(如以照像或数码信息的形式)转移到一个HIS系统或LIMS系统或电脑系统。
在上述本发明的组合体的一个实施方案中，毛细管中出液口及以上部位装有上述混合液，即样本例如尿液和能够与微孔膜和错误折叠蛋白竞争结合的染料进行混合形成的混合液体；优选地，所述混合液的体积为1～30μL，更优选，为1～25μL，例如大约1到5、6、7、8、9、10、11、12、13、14、15或16μL；更优选地8～15μL。
进一步地，在上述实施方案中，由于毛细管与微孔膜紧密接触并密合，毛细管中所含有一定量的混合液缓慢释放到微孔膜上，由此在毛细管的出液口周围的微孔膜表面形成上述扩散斑点。扩散斑点的示例包括图15中所示；在一个具体实施方案中，混合液从毛细管中释放出来后染料在出液口聚集、不扩散并形成深色斑点；在另一个具体实施方案中，混合液从毛细管中释放出来后染料在出液口附近形成深色斑点，还有“伪足”状扩散；在另一个具体实施方案中， 混合液从毛细管中释放出来后染料在出液口附近形成浅色扩散但包含伪足。在另一个具体实施方案中，混合液从毛细管中释放出来后，在出液口形成较小的红色斑点，但周围形成明显的圆形扩散斑点，或点样部位形成向外扩散的不规则扩散斑点，但没有“伪足”，或形成均匀的、较大的圆形扩散斑点。
在本发明的检测方法一个优选实施方案中，检测样本中是否含有错误折叠蛋白质或错误折叠蛋白质聚集物的方法包括以下步骤：
将样本和能够与微孔膜和错误折叠蛋白竞争结合的染料进行混合形成第一混合液；
用第一毛细管吸取一定量的第一混合液，并将第一毛细管的出液口与第一微孔膜紧密接触从而使第一毛细管中的第一混合液缓慢释放到第一微孔膜上；
用第二毛细管吸取一定量的阴性对照溶液即第二混合液，并将第二毛细管的出液口与第二微孔膜紧密接触从而使第二毛细管中的第二混合液缓慢释放到第二微孔膜上；
用第三毛细管吸取一定量的阳性对照溶液即第三混合液，并将第三毛细管的出液口与第三微孔膜紧密接触从而使第三毛细管中的第三混合液缓慢释放到第三微孔膜上。
进一步地，基于染料的颜色，观察第一、二、三混合液在微孔膜上的扩散情况以判定样本中含有错误折叠蛋白质或错误折叠蛋白质聚集物。
其中第二混合液可以是确定为不含错误折叠蛋白质的染料溶液，或者是确定为不含错误折叠蛋白质的生物样本例如阴性尿液与染料的混合液。其中第三混合液可以是确定为含错误折叠蛋白质的染料溶液，可以通过将确定为含错误折叠蛋白质的阳性样本与染料混合形成，或者通过将错误折叠蛋白质阳性参考品与染料混合而形成。在优选方案中，上述方法可以同时检测多个(2、3、4、5、10个或更多)样本，即将这些多个样本分别与染料混合分别形成混合液，然后分别用毛细管点样形成扩散斑点，并根据扩散斑点判定这些样本是否含有错误折叠蛋白质。
类似地，本发明的组合体中也可以包括2、3、4、5、6、7、8、9、10个、12个或更多毛细管，每个毛细管的出液口都分别与微孔膜的表面接触并密合， 这些毛细管可以通过连接桥固定在一起，形成联排毛细管构成的点样器。如图16所示，在图16A中，组合体160A包括毛细管162、182和192各自分别有一个出液口164、184、和194以及另一开放的管口166、186、196，开放的管口166、186、196穿透连接桥，从而毛细管两端均与外界连通。连接桥180作为支撑和固定毛细管162、182和192，组合体160A出液口164、184、和194分别与微孔膜168、188、198的表面接触并密合。如图16A所示，毛细管162、182和192各装有混合液，优选地，毛细管162中的液体是待测孕妇尿液样本和染料的混合液，毛细管182中的液体是阴性对照样本或缓冲液和染料的混合液，毛细管192中的液体是阳性对照样本和染料的混合液。
图16B显示上述图16A的组合体的毛细管中的液体部分被微孔膜吸收；图16C显示上述图16A的组合体的毛细管中的液体全部被微孔膜吸收，在微孔膜中形成不同的斑点扩散结果。
检测装置
本发明的另一方面提供一种检测样本中是否含有错误折叠蛋白质或错误折叠蛋白质聚集物的装置。
综合并引用上述说明及图12、13、14，本检测装置发明的一个具体实施方案可以包括一个带有两排凹槽的面板和一个嵌入面板中并可从面板上拆卸下来的点样部件，其中面板上的两排凹槽中，每排有1到多个(3个、10个、12个或更多)凹槽，其中一排为样本槽，另一排为测试槽。样本槽的直径为0.3～1cm，最优选为0.5cm，深度为0.3～1cm，最优选为0.5cm；样本槽可以装盛30-500μL或更多液体，优选地，面板上有3个以上样本槽包括一个待测样本槽、一个阴性对照样本槽、一个阳性对照样本槽。待测样本槽可以装有上述染料或染料溶液或染料与待测样本的混合液；阴性对照样本槽可以装有阴性对照样本；阳性对照样本槽可以装有阳性对照样本。样本槽可以用密封材料封口。测试槽的直径为0.5～2cm，最优选为1.8cm，深度为0.2～1cm，最优选为0.3cm。测试槽的底部覆盖有上述微孔膜如滤纸。一般来说，面板上的测试槽数量与样本槽一致或更多。嵌入面板中并可从面板上拆卸下来的点样部件如下所述，具体地，包含 一个或多个毛细管，毛细管可固定在一个横梁或柱状体上，例如，同样长度的2个或多个毛细管平行、并排地固定于一个横梁上，并且毛细管间的间距与样本槽间以及测试槽间的间距相对应以至于点样部件上的多个毛细管可同时插入相应的多个样本槽或者相应的多个测试槽。
例如，图2显示的本发明的检测装置包括测试部件和点样部件，测试部件包含微孔膜，点样部件包含毛细管，优选地，毛细管的数目为2个以上，例如2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个。
在本发明的一些实施方案中，测试部件可以是单独的微孔膜。
在本发明的另一些实施方案中，测试部件由第一盖板和第一底板组成，第一盖板和第一底板或拆卸或不可拆卸地连接。在本发明的一些实施方案中，第一盖板和第一底板不可拆卸地连接，从而成为一个整体，虽然物理上不再区别第一盖板和第一底板，但为了描述的方便，仍这样区分。
在本发明的一些具体实施方案中，第一盖板上覆盖有微孔膜，如图4A、B所示的那样。任选地，微孔膜为1个或多个，例如2个、3个、4个、5个、10个、20个、50个、100个、200个，微孔膜可为任意形状，例如圆形、椭圆形、方形或不规则形状。任选地，1个微孔膜只能供1个样本扩散，更优选地，微孔膜为圆形，其面积不小于样本所能扩散的最大面积。任选地，1个微孔膜可供多个样本扩散，如2个、3个、4个、5个、10个、20个、50个、100个、200个或更多，更优选地，微孔膜可分成多个区间，每个区间里可供一个样本扩散，更优选地，区间为正方形，其内切圆面积不小于样本所能扩散的最大面积。
在本发明的一个具体实施方案中，第一盖板上设有测试槽，测试槽是圆柱形凹槽，深度为载样部件厚度的1/3～2/3，凹槽中设有微孔膜，微孔膜为圆形，直径略小于凹槽横截面直径，平铺在凹槽底部，如图4C、D所示的那样。在本发明的具体实施方案中，测试槽的数目为多个，如为2个、3个、5个、10个、15个、20个、30个、50个、100个、200个，优选地，每个测试槽供一个样本进行扩散。
在本发明的另一些实施方案中，第一盖板和第一底板可拆卸地连接，任选地，为通过在边线和/或四角设置卡扣连接，任选地，通过粘合剂进行粘合连接， 任选地，通过焊接连接。在本发明的具体实施方案中，第一盖板上设有圆孔，第一盖板和第一底板之间设置微孔膜，当第一盖板和第一底板扣合或卡合或粘合后，第一盖板上的圆孔就成为凹槽，凹槽底部即为微孔膜，从而形成测试槽，如图4E、F所示的那样。
在本发明的一个实施方案中，检测装置还包括载样部件，由第二盖板和第二底板组成，其中第二盖板上设置样本槽，样本槽为圆柱形凹槽。
在本发明的优选实施方案中，第二盖板的下表面可拆卸或不可拆卸地与第二底板的上表面连接；优选为可拆卸连接，更优选为通过在边线和/或四角设置卡扣连接，或者通过粘合剂进行粘合连接，或者通过焊接连接。
在本发明的一个实施方案中，第一盖板的侧面不可拆卸地与第二盖板的侧面连接，成为一个整体，物理上没有区别，并且第一底板的侧面不可拆卸地与第二底板22的侧面连接，成为一个整体，物理上没有区别。
在本发明的一个实施方案中，第一盖板与第二盖板之间设置导流槽，该导流槽为与第一盖板和第二盖板中心连接垂直的长凹槽，凹槽横截面的形状为倒三角或圆弧状，设置导流槽的目的是防止样本槽中的液体流出，进入测试槽中，以产生检测结果偏差。有了导流槽，样本如果从样本槽中溢出或溅出，可以顺着第二盖板表面进入到导流槽中，储存在导流槽中，或顺着导流槽流出，而不再进入第一盖板。进一步地，在当样本槽数目多于一个时，在相邻两个样本槽中间同样可以设置导流槽，防止样本间交叉污染。
在本发明的另一个实施方案中，第一盖板11的侧面可拆卸地与第二盖板的侧面连接，或者第二底板的侧面可拆卸地第二底板的侧面连接。
在本发明的另一个实施方案中，第一盖板的侧面可拆卸地与第二盖板的侧面连接，并且第一底板的侧面可拆卸地第二底板的侧面连接。
在本发明一个更优选的实施方案中，连接是指通过活动部件5连接，例如通过软质材料相连，或者通过铰链相连，由此，载样部件可以与测试部件折叠。
在本发明的一个实施方案中，载样部件或测试部件的形状可以为任意形状，优选为选自三角形、四边形、多边形、椭圆形和圆形中的一种，更优选地，载样部件或测试部件的形状呈轴对称。
在本发明的一个具体实施方案中，样本槽的数目与测试槽的数目相同；
在本发明的一个实施方案中，样本槽和测试槽为单排直线排列、多排直线排列、或呈多边形排列、圆形排列、任意排列中的一种，优选地，呈直线排列。
在本发明的另一个具体实施方案中，样本槽和测试槽排列方式相同。
在本发明的一些实施方案中，样本槽和/或测试槽可以为任意形状，优选为选自三角形、四边形、多边形、椭圆形和圆形中的一种。
在本发明的一个具体实施方案中，样本槽和测试槽为圆形。
在本发明的一些实施方案中，样本槽的直径为0.3～1cm，最优选为0.5cm，深度为0.3～1cm，最优选为0.5cm。
在本发明的一些实施方案中，测试槽的直径为0.5～2cm，最优选为1.8cm，深度为0.2～1cm，最优选为0.3cm。
在本发明的一个具体实施方案中，样本槽和测试槽大小相同。
在本发明的一个实施方案中，点样部件为一空心盒状部件，其中含有毛细管。
在本发明的一些具体实施方案中，空心盒状部件与载样部件和/或测试部件可拆卸或不可拆卸地连接，空心盒装部件有一个开口，方便取放毛细管，优选地，开口与样本槽和测试槽开口朝向相同，更优选地，开口设置盖子。
在本发明的一些实施方案中，点样部件为一实心盒状部件，其上设有凹孔，用于放置毛细管。
在本发明的一些具体实施方案中，实心盒状部件与载样部件和/或测试部件可拆卸或不可拆卸地连接。优选地，实心盒状部件与载样部件和/或测试部件为一个整体。更优选地，直接在载样部件和/或测试部件上设置凹孔，用于放置毛细管，优选地，凹孔为一个，可容纳所有毛细管；优选地，凹孔可放置多个毛细管，更优选地，凹孔数目与毛细管数目相同，凹孔直径和深度均略大于毛细管，每个凹孔放置一个毛细管。凹孔不与样本槽或测试槽交叉。
在本发明的一个实施方案中，点样部件为点样器，其侧面设有毛细管。点样器主体为一长方体，长方体侧面设有凹孔，凹孔贯穿长方体，从而毛细管两端均与外界相通，毛细管插入到凹孔中，在本发明的一个具体实施方案中，毛 细管与长方体为一个整体。对应地，载样部件和/或测试部件1设置凹孔，点样部件的毛细管突出部分可以插入到对应的凹孔中。
在发明的一个具体实施方案中，毛细管的数目与样本槽数目或测试槽数目相同，排列方式与样本槽或测试槽相同。在本发明的一个具体实施方案中，毛细管为2个或2个以上，例如为3个，设置在长方体主体同一侧，使得点样部件呈现联排毛细管形状。
在本发明的一个具体实施方案中，点样器设置毛细管的侧面还包括定位针，第一盖板上设置定位孔，第二盖板上设置定位孔，当定位针插入到第二盖板的定位孔时，毛细管插入到样本槽中吸取液体，当定位针插入到第一盖板的定位孔时，毛细管接触测试槽中的微孔膜，毛细管中的液体被微孔膜吸附并在微孔膜上进行扩散。
在本发明的一个实施方案中，定位针的形状选自子弹头形状、圆柱、矩形柱、三棱柱、不规则柱、任意柱状或倾斜的任意柱状中的一种。在本发明的一个具体实施方案中，定位针的形状为子弹头形状。
在本发明的一个实施方案中，定位针为中空结构。
在本发明的一个实施方案中，定位针的位置在点样器的两侧或毛细管的中间。
在本发明的一个实施方案中，定位针的数目至少为2个，优选地为2个、3个、4个或更多。
在本发明的一个具体实施方案中，定位针的数目为2个，分别设置在点样器的两侧。
在本发明的一个具体实施方案中，载样部件和/或测试部件的不同外表面上还设置凹孔，凹孔排列方式与点样器上毛细管和定位针排列方式相同，从而可以将点样器固定在载样部件或测试部件上。
在本发明的一个具体实施方案中，点样器上设有凸起或凹槽，目的是增加摩擦力，方便拿捏、插入或拔出点样器。
在本发明的一些实施方案中，毛细管一端连接可使毛细管内部产生负压的装置。在本发明的一个实施方案中，能够使毛细管内部产生负压的装置为橡胶 球，类似滴管那样，在使用时，捏住橡胶球，同时将毛细管的另一端放入液体样本中，松开橡胶球，橡胶球恢复形变，使得毛细管中产生负压，从而使得液体样本更快进入毛细管。在本发明的另一个优选实施例中，能够使毛细管内部产生负压的装置为活塞装置，如注射器、移液枪等。在使用时，先将毛细管另一端放入液体中，通过人工拉动活塞或者依靠弹性装置拉动活塞，使得毛细管内部产生负压，使得液体样本更快进入毛细管。在本发明的实施方案中，上述使得毛细管内部产生负压的装置同样能够使得毛细管内部产生正压，从而推动毛细管中的液体缓慢流出，转移至微孔膜上，完成检测。
在本发明的一些实施方案中，载样部件、测试部件和点样部件采用PVC材料、PUC材料、尼龙材料、橡胶材料、丙烯腈-丁二烯-苯乙烯共聚物材料(ABS材料)、玻璃材料或金属材料制成。优选地，金属材料为合金材料，更优选地，合金材料为不锈钢。
本发明的检测装置至少有2个核心部件，包括微孔膜的检测部件和包括毛细管的点样部件。因此，本领域内的技术人员根据本说明书公开的内容就可明了其制备方法：对于检测部件，可将微孔膜固定在支持表面，可通过图4所示的3种方法，也可以选择其它方法。对于点样部件，如果毛细管多于1个，例如3个，可利用连接桥将毛细管连接，如图1C所示，毛细管包括出液口和另一开放口，贯穿连接桥，使得另一开放口也与空气连通。毛细管的排列方式可以是其他任意方式。
试剂盒
本发明另一方面提供一种检测样本中是否含有错误折叠蛋白试剂盒，包括微孔膜和毛细管。
本发明的一些实施方案中，试剂盒包括以上的任一检测装置。
在一个具体的实施方案中，试剂盒中的检测装置包括一个带有至少两排凹槽的面板和一个嵌入面板中并可从面板上拆卸下来的点样部件，其中面板上的两排凹槽中，每排有1到多个(3个、10个、12个或更多)凹槽，其中一排为样本槽，另一排为测试槽。优选地，面板上有3个以上样本槽包括一个待测样本 槽(装有上述染料或染料溶液或染料与待测样本的30-500μL或更大体积的混合液)、一个阴性对照样本槽(装有至少30μL阴性对照样本)、一个阳性对照样本槽(装有至少30μL阳性对照样本)。样本槽用密封材料封口。测试槽的底部覆盖有上述微孔膜如滤纸。嵌入面板中并可从面板上拆卸下来的点样部件包含一个或多个毛细管，同样长度的2个或多个毛细管平行、并排地固定于一个横梁上，并且毛细管间的间距与样本槽间以及测试槽间的间距相对应以至于点样部件上的多个毛细管可同时插入相应的多个样本槽吸取其中液体，然后同时插入相应的多个测试槽。
在本发明的具体实施方案中，试剂盒包括能够结合微孔膜和错误折叠蛋白的染料。染料选自杂环染料、刚果红、硫磺素、伊文思蓝中的一种或多种，优选为刚果红。在本发明的优选实施方案中，染料以固体干粉或溶液的形式预先装入样本槽中。
在本发明的具体实施方案中，试剂盒还包括对照样本，对照样本包括阳性对照样本和/或阴性对照样本。在本发明的优选实施方案中，对照样本预先装入对照样本槽中。
在本发明的具体实施方案中，样本槽利用密封材料封口，密封材料选自锡纸、塑料薄膜、铝箔。封口可以通过粘胶或塑封或其它任何能够密封的方法。
在本发明的一个具体实施方案中，阴性对照样本不含错误折叠蛋白，阳性对照样本含有错误折叠蛋白。在本发明的一些具体实施方案中，阳性对照样本包含任何与错误折叠蛋白质具有相同或相近结构，具有能够与微孔膜竞争结合染料(如刚果红)特性的物质，例如具有β片层结构的蛋白质，具体如经过变性处理的牛血清白蛋白(BSA)。在本发明的一个具体实施方案中，阴性对照样本为PBS缓冲液，阳性对照样本是包含错误折叠BSA的PBS缓冲液。在本发明的另一个具体实施方案中，阴性对照样本为不包含错误折叠质的尿液样本，阳性对照为包含错误折叠蛋白质的尿液样本。
在一些实施方案中，试剂盒还可以包含上述比对卡、干燥剂、说明书。在本发明的具体实施方案中，样本来自具有错误折叠蛋白疾病的病人体液，包括但不限于全血、血清、血浆、尿液、唾液、汗液、脑脊液、胸腹水、泪液、阴 道分泌物、精液、组织裂解液及其组合。当样本含有血液而呈颜色，可以在检测前进行离心去处红细胞或其它干扰因素。在本发明的一个具体实施方案中，错误折叠蛋白疾病为子痫前期，样本为孕妇尿液。
为了使本发明所解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例
实施例1
本实施例提供本发明所使用的毛细管类型。如图1A所示，本发明所使用的毛细管外观可以是多种形状(图1A)。其与微孔膜接触一端(即出液口一端)的横截面也可以是多种形状，包括但不限于圆形、椭圆形、正多边形、不规则多边形、带有圆角的多边形(图1B)。这些毛细管均可以吸取5-20μL液体，当毛细管中含有液体，出液口一端与滤纸接触后，液体释放到滤纸上的速度不大于4μL/秒。
如果同时需要使用多个毛细管，例如3个，可制作联排毛细管。如图1C所示，利用连接桥108将毛细管连接，毛细管100的出液口102用于吸取液体和接触滤纸，另一开放口106贯穿连接桥108后与空气连通。
实施例2
本实施例提供一种试剂盒，包括图2、图3、图4的装置，还包括1个或2个内径为1.25mm的不锈钢毛细管。
图2所示的装置包括测试部件201，测试部件201包括滤纸251。滤纸在测试部件上有3种设置方式：
第一种：测试部件201为一长方体，长方体上表面覆盖一个圆形滤纸251(设置方式如图4A、B)。
第二种：测试部件201为一长方体，长方体上表面设有一个圆柱形凹槽231，凹槽底部设有滤纸251(如图4C、D)。
第三种：测试部件201包括盖板211和底板212，盖板上设有圆孔271。当盖 板211与底板212粘合时，中间设置一层滤纸251(如图4E、F)，这样，盖板211和底板212通过粘合后，圆孔形成测试槽231，测试槽底部即为滤纸251。
图3所示的装置包括测试部件301，测试部件301为一长方体，其中一个侧面上设有2个测试槽311和312，测试槽311和312各为一个圆柱形凹槽，凹槽深度约为长方体厚度的2/3，凹槽中设有滤纸，滤纸为圆形，略小于凹槽横截面，平铺在凹槽底部(设置方式如图4C、D所示，图3中未显示滤纸)。
用于检测孕妇尿液中是否含有错误折叠蛋白质的具体检测步骤为，将取自孕妇尿液的样本与刚果红混合得到混合液，将毛细管竖直放置在混合液中10秒左右(可吸取10μL左右混合液)，转移至测试槽，将毛细管的出液口与滤纸紧密接触，保持竖直，停留15秒左右，毛细管中的液体缓慢释放至滤纸上，移去毛细管，观察结果。
利用图2的装置只能检测1个待测样本。利用图3的装置可以检测2个待测样本，或者检测1个待测样本，1个对照样本，对照样本可以是阴性对照样本，也可以是阳性对照样本。阴性对照样本是PBS缓冲液，阳性对照样本为含有经过特殊变性处理形成错误折叠BSA的PBS缓冲液。
实施例3
本实施例提供一种试剂盒及使用方法，该试剂盒包括图5、图6、图7或图8所示的装置。
图5所示检测装置包括测试部件501和载样部件502，测试部件由第一盖板511和第一底板512组成，第一盖板511上设有3个圆柱形凹槽，即为测试槽514、516、518，测试槽底部按图4C、D设置滤纸534、536、538。第一盖板511的下表面不可拆卸地与第一底板512的上表面连接，成为一个整体。载样部件由第二盖板521和第二底板522组成，第二盖板上设有3个样本槽525、527、529。第二盖板521的下表面不可拆卸地与第二底板522的上表面连接，成为一个整体；第一盖板511的侧面与第二盖板521的侧面直接连接，成为一个整体；第一底板512的侧面与第二底板522的侧面直接连接，成为一个整体。由此，测试部件和载样部件成为一个整体，物理上没有任何分别。
图6所示的检测装置包括测试部件601和载样部件602，测试部件601由第一 盖板611和第一底板612组成，第一盖板611上表面设有5个测试槽631、633、635、637、639，测试槽底部分别设有滤纸651、653、655、657、659(设置方式如图4C、D所示)。载样部件由第二盖板621和第二底板622组成，第二盖板上表面设有5个样本槽641、643、645、647、649。第一盖板611的侧面通过软质材料675与第二盖板621的侧面连接，第一底板612的侧面与第二底板622的侧面没有连接。由此，测试部件601和载样部件602在运输、储藏过程中可以折叠在一起，从而节省空间。
图7所示的检测装置包括测试部件701和载样部件702，测试部件上表面上设有3个测试槽731、733、735，测试槽底部分别设有滤纸751、752、753(设置方式如图4C、D所示)。载样部件702的上表面设有3个样本槽741、743、745。测试部件701和载样部件702通过活动铰链705连接。
图8所示的检测装置与图7基本相同，不同的是测试槽和样本槽的数目比图7所示的要多(14个)，这样可以用来检测更多的样本。
任一试剂盒还包括与样本槽数目相同的，内径为1.25mm的不锈钢毛细管。
用于检测孕妇尿液中是否含有错误折叠蛋白质的具体检测步骤为，将取自孕妇尿液的样本与刚果红(浓度5mg/mL)以20：1的比例在样本槽中混合得到混合液，竖直放置在混合液中10秒左右(可吸取10μL左右混合液)，将毛细管转移至测试槽，使其出液口与滤纸接触，停留15秒左右，毛细管中的液体缓慢释放至滤纸上，移去毛细管，观察结果。
利用图5和图7所示的装置最多可以检测3个待测样本，当然，也可以检测1个待测样本，另外两个测试槽用于检测阴性对照样本和阳性对照样本。检测方法与此相同。利用图6的装置最多可以检测5个待测样本，或者检测3个待测样本、2个对照样本即阴性对照样本和阳性对照样本。利用图8所示的装置最多可以检测14个待测样本，或者检测12个待测样本，1个阴性对照样本，1个阳性对照样本。
实施例4
本实施例提供一种试剂盒及使用方法，该试剂盒包括图9、图10或图11所示的装置。
图9所示的检测装置包括测试部件901、载样部件902和点样部件903。测试部件由第一盖板911和第一底板912(图中未示出)组成，第一盖板911上设有14个测试槽931。载样部件902由第二盖板921和第二底板922(图中未示出)组成，第二盖板921上设有14个样本槽941。第一盖板911和第一底板912为一个整体，物理上不可区分，测试槽931中设有滤纸951，按图4C、D所示方式设置。第二盖板921与第二底板922为一个整体，物理上不可区分。测试部件901和载样部件902通过卡扣905连接在一起，运输储藏时可分开叠放在一起，使用时连接在一起。点样部件903为一空心盒状部件，其中包含毛细管933，该毛细管933为内径为1.2mm的玻璃管，空心盒状部件与载样部件902和测试部件901通过卡扣连接。
图10所示的检测装置包括测试部件1001、载样部件1002和点样部件1003。测试部件由第一盖板1011和第一底板1012组成，第一盖板1011上设有5个测试槽1031。载样部件由第二盖板1021和第二底板1022组成，第二盖板1021上设有5个样本槽1041。第一盖板1011与第一底板1012为一个整体，物理上不可区分，测试槽1031中按图4C、D方式设置滤纸1051。第二盖板1021与第二底板1022为一个整体，物理上不可区分。同样，第一盖板1011与第二盖板1021为一个整体，并且第一底板1012与第二底板1022为一个整体。也就是说，测试部件1001和载样部件1002为一个整体，只是在划分上进行区别。点样部件1003为一实心盒状部件，其上设置与样本槽数目同样多的凹孔1032，所述凹孔1032的直径略大于毛细管(图中未示出)直径，深度略大于毛细管长度，每个凹孔可放置一个毛细管。该实心盒状部件上表面与第一底板1012下表面和第二底板1022下表面不可拆卸地连接，并成为一个整体。
图11所示的检测装置，包括括测试部件1101、载样部件1102和点样部件1103。测试部件由第一盖板1111和第一底板1112组成，第一盖板1111上设有5个测试槽1131；载样部件由第二盖板1121和第二底板1122组成，第二盖板1121上设有5个样本槽1141。同图10所示的检测装置一样，该检测装置为一个整体，只是划分上进行区别，并且第一盖板1111上设有测试槽1131，同样按照图4C、D的方法放置滤纸。不同的是，点样部件1103位于测试部件1101和载样部件1102 侧面。点样部件1103上设置凹孔1151，可容纳不少于样本槽数目的毛细管1153(内径为1.25mm的不锈钢毛细管)，深度略大于毛细管1153长度(3cm)。
使用步骤同实施例2、3。
实施例5
本实施例提供一种试剂盒及使用方法，该试剂盒包括图12、图13和图14所示的装置。
图12所示的检测装置，包括测试部件1201、载样部件1202和点样部件1203，所有部件由PVC材料制成。测试部件由第一盖板1211和第一底板1212(图中未示出)组成。载样部件由第二盖板1221和第二底板1222(图中未示出)组成，第二盖板1221上设有3个样本槽1241。第一盖板1211的侧面不可拆卸地第二盖板1221的侧面连接，成为一个整体，并且第一盖板1211和第二盖板1221之间设置导流槽1206，导流槽1206与测试部件1201和载体部件1202中心连线垂直，横截面呈倒三角形状，贯穿整个上表面。第一底板1212的侧面不可拆卸地第二底板1222的侧面连接，成为一个整体。第一盖板1211上设有3个圆孔，在与第一底板1212连接时，中间放置一层滤纸(如图4E、F)，这样，第一盖板1211和第一底板1212通过四角卡扣盖合后，圆孔形成测试槽1231，测试槽底部即为滤纸1251。
点样部件1203为点样板，该点样板的侧面设有毛细管1233，毛细管的数目与样本槽数目或测试槽数目相同，即都为3个，排列方式与样本槽或测试槽相同；点样板设置毛细管的同一侧面，毛细管的两侧还包括定位针1235，第一盖板1211和第二盖板1221上设置定位孔1204和1208，当定位针1235插入到定位孔1204或1208中时，毛细管1233可插入样本槽1241或测试槽1231中。载样部件1202相对于测试部件1201的侧面还设置凹孔(图中未示出)，凹孔排列方式与点样板上毛细管1233和定位针1235排列方式相同，从而可以将点样部件1203固定在载样部件1202上。点样板上还设置凸起1237，方便将点样板插入拔出。
在样本槽中，预先加入刚果红，其中一个样本槽还加入阳性对照样本(含有BSA的PBS缓冲液)，另一个样本槽中加入阴性对照样本(不含有BSA的PBS缓冲液)，并利用锡纸密封。
图13所示的检测装置与图12所示的检测装置基本相同，不同的是测试槽和样本槽的数目比图12所示的要多(5个)，这样可以用来检测更多的样本。
本试剂盒的使用方法如下：
1、将点样部件拔出，并将检测装置主体(包装测试部件和载样部件)水平放置于台面上；
2、利用定位针依次将样本槽上的锡纸刺破(图14A)；
3、在待测样本槽中加入孕妇尿液；
4、将点样部件上的定位针插入到第二盖板的定位孔中(图14B)，由此点样部件上的毛细管插入到样本槽中的液体中，从而液体会自动毛细管中上升；
5、将点样部件的定位针插入到第一盖板的定位孔中(图14C)，由此点样部件上的毛细管直接接触位于测试槽中的微孔膜上(图14D剖面图所示)，静置5-10秒；
6、移去点样部件，观察检测结果。
7、若结果如图15A所示，即点样部位显示较为聚集、不扩散的红色斑点(图15A，a)，或者除了点样部件显示较为聚集、不扩散的红色斑点，还有“伪足”(图15A，b)，或者还伴随轻微的扩散(图15A，c)，则孕妇尿液样本判定为阴性，即不含有错误折叠蛋白质或含有的错误折叠蛋白质低于参考值。
若结果如图15B所示，即点样部位显示较小红色斑点，但周围有较为明显的扩散(图15B，d)，或点样部位显示红色斑点，具有不规则扩散的浅绝色圆形(图15B，e)，或形成均匀的、较大的扩散斑点(图15B，f)，则孕妇尿液样本判定为阳性，即含有错误折叠蛋白质或含有的错误折叠蛋白质高于参考值。
本实施例提供的检测装置将器材和试剂集成在一起，大大压缩了包装空间，方便运输、存储，且便于实验室操作。
实施例6
本实施例提供一种简便试剂盒及使用方法，该试剂盒包括滤纸和毛细管 (内径为1.25mm的不锈钢毛细管)。使用方法如下：
1、将滤纸水平放置于台面上；
2、将取自孕妇的尿液样本与刚果红染料混合成混合液；
3、取毛细管吸取混合液，转移至滤纸上紧密接触并密合，停留5秒以上使得毛细管中液体被缓慢、充分释放在滤纸上；
4、静置10秒左右，即可观察检测结果。
图16示出了本发明检测装置检测过程示意图，毛细管接触滤纸后，样本缓慢释放在滤纸上，并在接触位置向外扩散，形成不同大小的染色斑点。
实施例7
为了比较毛细管和移液枪或滴管点样对检测结果的影响，发明人设计以下实验：
1.毛细管、移液枪和滴管
采用的毛细管是圆形出液口内径为1.25mm、长度为10cm的玻璃毛细管，毛细管上标有10μL刻度线。
采用的移液枪为10μL的移液枪。
采用的滴管为100μL，每次测试吸取10μL液体。
2.阴性样本和阳性样本
阴性样本为医生根据临床结果判断为非子痫前期的孕妇尿液样本，阳性样本为医生根据临床结果判断为子痫前期的孕妇尿液样本。
3.检测
分别在阴性样本和阳性样本中按50：1的比例加入刚果红。将毛细管分别插入阴性样本或阳性样本中，待液体上升到10μL刻度线时，取出毛细管，将吸取液体的一头接触滤纸，待样本全部被滤纸吸收，移除毛细管，扩散15秒左右后，观察检测结果。
同样，利用移液枪和滴管分别吸取10μL阴性样本或阳性样本，之后将移液枪枪头或滴管滴头与滤纸接触，释放出吸取的液体至滤纸上，扩散一段时间后，观察检测结果。
为了比较接触点样和悬空点样对检测效果的区别，同样分别利用毛细管、 移液枪和滴管吸取10μL阴性样本或阳性样本，将毛细管出液口、移液枪枪头或滴管滴头悬空在滤纸上方0.5～2cm处，释放出吸取的混合液至滤纸上，扩散一段时间后，观察检测结果。
分别测量阴性样本和阳性样本扩散斑点半径，扩散斑点半径为点样中心点到斑点扩散至最远位置的长度。并计算阳性样本扩散斑点半径与阴性样本扩散半径的半径比。每个实验重复5次。
4.结果
利用毛细管、移液枪和滴管点样的斑点扩散情况如图17或表1所示。对于阳性样本：
不管哪种点样器，也不管哪种点样方式，阳性样本均会形成均匀的、较大的扩散斑点，几乎没有区别。
对于阴性样本：
三种点样器(毛细管、移液枪和滴管)悬空点样时，阴性样本均形成相对较大的斑点，与阳性样本同样的点样方式形成的斑点的半径比在1.2左右，区别相对较小，容易形成误判。
而当接触点样时，毛细管检测形成的斑点较为聚集、不向外扩散或极少扩散，与阳性样本形成的斑点半径比在2.5以上。移液枪和滴管接触点样，阴性样本形成的扩散斑点斑点相对较大，且有不规则向外扩散，半径比在1.6左右。
表1 利用毛细管、移液枪和滴管点样对斑点扩散的影响(n＝5，p<0.01)
Figure PCTCN2018115444-appb-000001
综上所述，利用毛细管接触点样检测效果最好，区分更明显，不易形成误判，结果直观可靠。
实施例8
为了比较毛细管不同内径对检测结果的影响，发明人设计以下实验：
1.毛细管
采用的毛细管为内径分别为0.4、0.5、0.6、0.7、0.9、1.12、1.25、1.45、1.69、1.99、2.4、2.64和2.8mm，长度为3cm的不锈钢毛细管，毛细管上标有10μL刻度线。
2.阴性样本和阳性样本
阴性样本为医生根据临床结果判断为非子痫前期的孕妇尿液样本，阳性样本为医生根据临床结果判断为子痫前期的孕妇尿液样本。
3.检测
分别在阴性样本和阳性样本中加入刚果红。将不同内径的毛细管分别插入阴性样本或阳性样本中，停留30秒以上，待液体不再上升时，取出毛细管，将吸取液体的一头接触滤纸，待样本全部被滤纸吸收(约3-10秒)，移除毛细管，扩散一段时间后，观察检测结果。
分别测量阴性样本和阳性样本扩散斑点半径，扩散斑点半径为点样中心点到斑点扩散至最远位置的长度。并计算阳性样本扩散斑点半径与阴性样本扩散半径的半径比。每个实验重复5次。
4.结果
利用不同内径毛细管点样的斑点扩散情况如图18或表2所示，可见当毛细管内径太小(例如小于0.7mm时)，液体从毛细管扩散到滤纸上的速度过慢，阳性样本的斑点扩散半径太小，与阴性样本的斑点扩散半径区别不大。随着毛细管内径的增加，阴性样本的斑点扩散增加不明显，但阳性样本的斑点扩散迅速增加。当毛细管内径增加到一定程度(例如大于3mm)，由于液体与滤纸的接触面积太大，阴性样本的斑点扩散半径也开始变大，但阳性样本的斑点扩散半径变化不明显，从而导致半径比不再增加甚至出现下降的趋势。当毛细管进一步增大时，液体不再能够自主进入毛细管中，导致无法进行检测。在本试验中，当毛细管内径0.7-2.8mm时检测效果较好，1.12-2.64mm时，检测效果更好。
表2 不同毛细管内径对斑点扩散的影响(n＝5，p<0.01)
Figure PCTCN2018115444-appb-000002
实施例9
为了比较不同点样量对检测结果的影响，发明人设计以下实验：
1.毛细管
采用的毛细管为内径1.25mm，长度为5cm的玻璃毛细管，该毛细管最大取液量为20μL，毛细管上标有范围为1-20μL，精度为1μL的刻度线。
2.阴性样本和阳性样本
阴性样本为医生根据临床结果判断为非子痫前期的孕妇尿液样本，阳性样本为医生根据临床结果判断为子痫前期的孕妇尿液样本。
3.检测
分别在阴性样本和阳性样本中加入刚果红。将毛细管分别插入阴性样本或阳性样本中，分别吸取2、3、4、5、6、7、8、9、10、11、12、13、14、15、16或17μL液体后，取出毛细管，将吸取液体的一头接触滤纸，待样本全部被滤 纸吸收，移除毛细管，扩散一段时间后，观察检测结果。
分别测量阴性样本和阳性样本扩散斑点半径，扩散斑点半径为点样中心点到斑点扩散至最远位置的长度。并计算阳性样本扩散斑点半径与阴性样本扩散半径的半径比。每个实验重复5次。
4.结果
不同点样量的斑点扩散情况如图19或表3所示，可见随着点样量的增加，阴性样本的斑点扩散半径增加不明显，但阳性样本的斑点扩散半径迅速增加，阴性样本和阳性样本的区分越来越明显，表现为半径比逐渐增大。但当点样量达到16μL时，阳性样本的斑点扩散半径增加变缓，甚至不再增加，这是受到扩散力学的影响，使得斑点扩散集中在一定的区域内。另一方面，随着点样量的增加，毛细管吸附时间越来越长，并且由于阴性样本由于不扩散或扩散程度很小，毛细管中的液体难以被彻底吸附至滤纸上或者需要更长时间才能完全被吸附，从而造成检测时间过长。综合考虑，点样量至少4μL，在5-15μL时效果较好。
表3 不同点样量对斑点扩散的影响(n＝5，p<0.01)
Figure PCTCN2018115444-appb-000003
Figure PCTCN2018115444-appb-000004
以上例子在此用于示范本发明的优选实施方案。本领域内的技术人员会明白，上述例子中披露的技术代表发明人发现的可以用于实施本发明的技术，因此可以视为实施本发明的优选方案。但是本领域内的技术人员根据本说明书应该明白，这里所公开的特定实施例可以做很多修改，仍然能得到相同的或者类似的结果，而非背离本发明的精神或范围。
除非另有定义，所有在此使用的技术和科学的术语，和本发明所属领域内的技术人员所通常理解的意思相同，在此公开引用及他们引用的材料都将以引用的方式被并入。
那些本领域内的技术人员将意识到或者通过常规试验就能了解许多这里所描述的发明的特定实施方案的许多等同技术。这些等同将被包含在权利要求书中。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种检测样本中是否含有错误折叠蛋白的装置，包括：
    具有面积不超过9mm 2的横截面的出液口、并且能够吸取和容纳至少5μL液体的毛细管；和
    能够与错误折叠蛋白质竞争结合染料的微孔膜，其中，所述毛细管的出液口与所述微孔膜的表面接触并密合。
  2. 根据权利要求1所述的装置，其中所述毛细管中包含所述染料和生物样本混合而成的至少5μL的混合液，其中所述染料选自杂环染料、刚果红、硫磺素、伊文思蓝中。
  3. 根据权利要求2所述的装置，其中所述毛细管出液口的周围的所述微孔膜的表面由于所述毛细管内所述混合液自所述毛细管出液口缓慢释放而染色。
  4. 一种检测样本中是否含有错误折叠蛋白的装置，包括：
    带有至少第一排凹槽和第二排凹槽的面板，以及嵌入面板中并可从面板上拆卸下来的点样部件，其中
    所述第一排凹槽包括一个待测样本槽、一个阴性对照样本槽、一个阳性对照样本槽分别可以装盛30-500μL或更多液体；
    所述第二排凹槽包括至少三个底部覆盖有能够与错误折叠蛋白质竞争结合染料的微孔膜的测试槽；
    所述点样部件包含同样长度至少三个相互平行的出液口横截面面积不超过9mm 2并且能够吸取和容纳至少5μL液体的毛细管并排地固定于一个横梁上，并且所述毛细管间的间距与所述样本槽间以及测试槽间的间距相对应以至于所述至少三个毛细管可同时分别插入相应的所述样本槽或者相应的所述测试槽。
  5. 根据权利要求4所述的装置，其中，所述待测样本槽装有能与错误折叠蛋白质特异性结合的染料或染料溶液；所述阴性对照样本槽装有所述阴性对 照样本；所述阳性对照样本槽装有所述阳性对照样本；所述样本槽用密封材料封口。
  6. 一种检测样本中是否含有错误折叠蛋白的试剂盒，包括
    出液口横截面的面积不超过9mm 2并且能够吸取和容纳至少5μL液体的毛细管；和
    能够与错误折叠蛋白质竞争结合染料的微孔膜。
  7. 根据权利要求6所述的试剂盒，还包括印有至少阴性和阳性样本扩散结果示例的比对卡。
  8. 一种检测样本中是否含有错误折叠蛋白的试剂盒，包括权利要求4或5的装置。
  9. 一种检测样本中是否含有错误折叠蛋白的检测系统，包括
    权利要求4或5的装置；
    信号采集模块包括光信号采集器；以及
    信息存储器用于储存所述信号采集模块采集到的经过处理或未经处理过的信号。
  10. 一种检测样本中是否含有错误折叠蛋白的方法，包括以下步骤：
    将样本和能够与微孔膜和错误折叠蛋白结合的染料进行混合形成混合液；
    用出液口横截面面积不超过9mm 2并且能够吸取和容纳至少5μL所述混合液的毛细管吸取至少4μL的所述混合液；
    将所述毛细管的所述出液口与所述微孔膜紧密接触使所述毛细管中的所述混合液缓慢释放到所述微孔膜上；
    基于所述染料的颜色，观察所述混合液在所述微孔膜上的扩散情况来判定样本中是否含有错误折叠蛋白。
PCT/CN2018/115444 2017-12-28 2018-11-14 一种检测错误折叠蛋白质的装置、试剂盒和方法 WO2019128506A1 (zh)

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