WO2017156910A1 - 一种离心分离检测方法及装置 - Google Patents

一种离心分离检测方法及装置 Download PDF

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Publication number
WO2017156910A1
WO2017156910A1 PCT/CN2016/086842 CN2016086842W WO2017156910A1 WO 2017156910 A1 WO2017156910 A1 WO 2017156910A1 CN 2016086842 W CN2016086842 W CN 2016086842W WO 2017156910 A1 WO2017156910 A1 WO 2017156910A1
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Prior art keywords
solid phase
film
centrifugal
detection
sample
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PCT/CN2016/086842
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English (en)
French (fr)
Inventor
刘凤鸣
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北京康华源科技发展有限公司
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Priority claimed from CN201610143694.3A external-priority patent/CN107192819A/zh
Priority claimed from CN201610330378.7A external-priority patent/CN107402299A/zh
Application filed by 北京康华源科技发展有限公司 filed Critical 北京康华源科技发展有限公司
Priority to EP16894072.4A priority Critical patent/EP3431990A4/en
Priority to JP2018547942A priority patent/JP2019507887A/ja
Publication of WO2017156910A1 publication Critical patent/WO2017156910A1/zh
Priority to US16/124,858 priority patent/US20180364227A1/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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/805—Haemoglobins; Myoglobins

Definitions

  • the invention relates to a centrifugal separation detection method and device, in particular to a centrifugal separation detection method and a centrifugal separation detection device, which belong to the field of immunodetection technology and instrument analysis, respectively.
  • Immunological detection technology is an experimental method for measuring antigens, antibodies, immune cells and chemical components designed by the principle of immunology. It is widely used in samples for human disease and health detection and health testing, as well as for environmental and pharmaceutical applications. Samples for analysis, food and industrial analysis. Commonly used are immune turbidity technology, solid phase enzyme immunoassay technology, chemiluminescence detection technology, immunofluorescence labeling technology, flow cytometry, colloidal gold technology.
  • Immune turbidity technology also known as immunoturbidimetric method, is a soluble antigen, an antibody specifically binds in the liquid phase, produces a complex of a certain size, forms the refraction or absorption of light, and determines the transmitted or scattered light after such refraction or absorption. As a unit of calculation, it is used for quantitative detection, but the detection sensitivity is low and it is not suitable for micro detection.
  • the solid phase enzyme immunoassay technology is based on the immobilization of antigen or antibody and the enzymatic labeling of antigen or antibody. The antigen or antibody bound to the surface of the solid phase carrier maintains its immunological activity, and the enzyme conjugate of the antigen or antibody retains its immunology.
  • the activity while retaining the activity of the enzyme, in the measurement, the test specimen (measured antibody or antigen) and the enzyme target antigen or antibody react with the antigen or antibody on the surface of the solid phase carrier in different steps, and has high sensitivity.
  • the linear response range is wide and easy to automate, but the long detection time limits its use.
  • Immunochemiluminescence detection technology is a highly sensitive micro and trace analysis technology. It has the advantages of convenient operation, high sensitivity, wide linear response range and easy automation. It is widely used in environmental, clinical, pharmaceutical analysis, food and In industrial analysis, it is also a solid phase separation method based on an antigen or an antibody, and a luminescent reagent labeling technique for an antigen or an antibody.
  • the object of the present invention is to provide a centrifugal separation detecting method, and another object of the present invention is to provide a centrifugal separation detecting device, which has the characteristics of high sensitivity and short detection time, and the centrifugal separation detecting device of the present invention has detection Fast, high sensitivity and easy to use.
  • the centrifugal separation detecting method comprises the steps of: driving a liquid phase through a solid phase film by using a centrifugal device, wherein the liquid phase comprises a sample to be inspected and a detecting phase containing the object to be tested, and the detecting phase is capable of The substance to be detected directly or indirectly forms a substance which specifically binds to the detection indicator; when the liquid phase flows through the solid phase film, it is combined with the specific conjugate of the test substance coated on the solid phase film to form a detection.
  • the detector detects the indirect fixation to the solid phase film by detection The amount of the agent, that is, the content of the test substance is detected.
  • the rotating member of the centrifugal device is of a planar type or inclined outward from the center.
  • a middle portion of a plane of a rotating component of the centrifugal device is provided: a storage device for the sample to be inspected and the detecting phase, the sample to be inspected and the detecting Phase injection device and drive device;
  • the outer edge of the plane of the rotating member of the centrifugal device is provided with a placement device of the solid phase film.
  • the injection proximal end of the solid phase membrane is connected to the liquid adsorption dispersion device, and the distal end of the injection is connected to the liquid adsorption device;
  • the liquid adsorption dispersion device adopts a solid phase material having rapid liquid dispersion characteristics, and the solid phase material is a poly-polyester cellulose film and/or a glass cellulose film, specifically, a colloidal gold-labeled adsorption film, a fluorescently labeled antibody.
  • An adsorption film, a chemiluminescent-labeled adsorption film, and/or a dispersion film; the liquid adsorption device employs a water-absorbent material, which is an absorbent paper and/or a water-absorbent gel.
  • the detector is disposed on one side or both sides of the solid phase film.
  • the rotation speed of the centrifugal device and the injection speed of the sampling device are both controlled by a program
  • the rotation speed of the centrifugal device is 200-10000r/min, specifically 500-5000r/min, 800-3000r/min or 800-2000r/min;
  • the time of centrifugation by the centrifugation device may be 1 to 5 min, specifically 1 min or 1 to 3 min.
  • the solid phase film is a nitrocellulose membrane, a polyvinylidene fluoride membrane, a nylon membrane, a DEAE cellulose membrane, and the solid phase membrane has a backing on one or both sides;
  • the detector includes any of the detectors for absorbance, fluorescence, chemiluminescence, and image color digital processing.
  • the polyvinylidene fluoride film is referred to as a PVDF film;
  • the DEAE cellulose film refers to a paper-like film prepared by introducing diethylaminoethyl (DEAE) into cellulose molecules, and is a weak base anion exchange material.
  • the analyte is an immunologically active protein or a substance that is immunoconjugated with a protein
  • the analyte-specific conjugate is an antigen or an antibody that specifically binds to the analyte
  • the detection indicator is at least one of a colloidal metal, a dye, a fluorescein, and a chemiluminescent substance.
  • the colloidal metal is at least one of colloidal gold, colloidal selenium and colloidal gold magnetic particles;
  • the fluorescein is fluorescein isothiocyanate (FITC for short), tetraethyl rhodamine, rhodamine tetramethyl isothiocyanate, phycoerythrin (abbreviated as PE), polydactin chlorophyll protein (PerCP for short) And at least one of propidium iodide (PI), allophycoerythrin (abbreviated as APC) and an anthraquinone compound, wherein the antimony compound is specifically cerium oxide;
  • the chemiluminescent substance is luminol and isoluminol and its derivatives, acridinium ester and decanoic acid amide, (gold alkane)-1,2-dioxyethane and its derivatives and terpyridine At least one of the cockroaches.
  • the composite is formed by any one of the following:
  • the analyte-specific conjugate comprises a primary intermediate phase that simultaneously forms a specific binding with the analyte and the detection phase;
  • the analyte-specific conjugate comprises a secondary intermediate phase that simultaneously forms a specific binding with the primary intermediate phase and the detection phase;
  • the primary intermediate phase and the secondary intermediate phase are each at least one of an antigen, an antibody, avidin, biotin, and the like having specific binding ability;
  • the method further includes the step of washing the solid phase membrane with a washing phase after the binding reaction, the washing phase being at least one of a phosphate buffer solution, a carbonate buffer solution, and a Tris buffer solution.
  • the centrifugal separation detection method of the invention is applied to the detection of the content of an immune product.
  • the centrifugal separation detecting device comprises a sample injection part, a solid phase film, a centrifugal device and a detector;
  • the centrifugal device includes a centrifugal rotor driven by a driving motor and a supporting base supported by the supporting base;
  • the injection component is disposed in a middle portion of the centrifugal rotor and is coupled to the centrifugal rotor;
  • the sample injection component includes a liquid phase storage device and a sample tube; and the liquid phase storage device is in communication with the sample tube ;
  • the solid phase film is disposed on the centrifugal rotor and connected to the sample tube;
  • the detector is disposed on one or both sides of the solid phase film.
  • the solid phase film is placed in a solid phase film placing device, and the solid phase film placing device is disposed on the centrifugal rotor;
  • the solid phase membrane and the centrifugal rotor are of a detachable structure.
  • the solid phase film placing device is a rotary movable fixing device and/or a groove-shaped pressing fixing device.
  • the solid phase membrane is fixed by a solid phase membrane holder selected from the group consisting of a solid phase membrane supporting film-like member, a lateral flow test strip buckle component, and a transparent At least one of the upper and lower embedded components.
  • the transparent upper and lower embedding member is such that the upper and lower sides of the solid phase film are covered with a hard transparent material, and the area of the hard transparent material on the side corresponding to the solid phase film is Greater than or equal to the area of the solid phase film.
  • the solid phase film is further provided with a liquid adsorption dispersing member in communication with the sampling tube at the end connected to the sampling tube, and the solid phase film is at the telecentric end of the centrifugal rotor
  • a liquid collecting member is provided in communication therewith, and the liquid adsorbing and dispersing member is in communication with the sample tube.
  • the sample introduction unit includes a sample pump that drives the liquid in the liquid phase storage device into the sample introduction tube.
  • the liquid phase storage device includes a sample storage device to be inspected and a detection phase storage device;
  • the sample storage device to be tested and the detection phase storage device are both in communication with the sample tube and are driven by the sample pump.
  • the liquid phase storage device further includes a cleaning liquid storage device that communicates with the sampling tube and is driven by the sampling pump.
  • the centrifugal rotor is of a flat type or inclined outward from the center;
  • the centrifugal device is provided with an outer casing.
  • the rotary movable type fixing device is a connecting device for columnar protrusions provided on the centrifugal rotor, and the solid phase film holder is provided with a connecting device with the columnar protrusions Matching hole-shaped parts.
  • the centrifugal device In the above-described centrifugal separation detecting device, the centrifugal device, the sample introduction member, and the detector are each provided with a program control device.
  • the sample tube and the solid phase film are detachably connected;
  • the liquid adsorption dispersion member is in detachable connection with the sample tube.
  • the material of the solid phase film is any one of a nitrocellulose membrane, a polyvinylidene fluoride membrane, a nylon membrane, and a DEAE cellulose membrane, and one or both sides of the solid phase membrane With a backing;
  • the liquid adsorption dispersion member comprises at least one of a colloidal gold labeled adsorption film, a fluorescently labeled antibody adsorption film, a chemiluminescent label adsorption film, a poly polyester fiber dispersion film, and a glass fiber dispersion film;
  • the detector includes any of a detector of absorbance, fluorescence, chemiluminescence, and image digital processing.
  • the liquid collecting device may be made of a water-absorbent material, specifically an absorbent paper and/or a water-absorbent gel, and the liquid collecting device may be a liquid collecting container.
  • the planar intermediate portion of the centrifugal rotor is provided with a hole and/or provided with a transparent member, and the hole and/or the transparent member directly expose the solid phase film to the Detector.
  • the immune product includes at least one of an antigen, an antibody, an immune cell, and a chemical component.
  • the present invention uses a centrifugal device to drive the liquid phase to be flowed and cleaned on the solid phase membrane, thereby improving the capture and binding ability of the analyte, reducing the background noise interference of the solid phase membrane, improving the detection sensitivity of the method, and realizing the present High sensitivity detection with detection reagents.
  • the present invention uses a centrifugal device to drive the liquid phase detected on the solid phase membrane, which changes the existing membrane detection technology by relying on natural flow and the liquid is reduced as the flow on the membrane is prolonged, and the liquid can be kept in the membrane.
  • the uniform flow on the surface ensures the uniformity of the binding of the analyte on the membrane, which can improve the detection accuracy.
  • the invention uses a centrifugal device to drive the detection liquid phase to flow on the solid phase membrane, keeps the liquid flowing uniformly on the membrane, shortens the detection time, and has the advantages of rapid detection.
  • the centrifugal device drives the liquid flow and the injection pump to be injected, and the operation steps are simple, and it is convenient to develop a more convenient miniaturized detection device.
  • multi-step and multi-step drive control is adopted, which involves the defect of transposition and movement of the detection sample, the detection phase and the reaction carrier.
  • the operation steps of the invention are simple and easy to realize automatic operation.
  • the method of the invention has the characteristics of high sensitivity, full quantification and automation, and has the detection technology of rapid detection and simple use of equipment; not only is convenient to use, waste of raw materials is reduced, but also work efficiency is obviously improved, and is applied to detection, analysis and separation. Many areas.
  • Fig. 1 is a schematic view showing a centrifugal separation detecting device in a first embodiment of the present invention.
  • Figure 2 is a schematic illustration of the additional components of Figure 1.
  • Fig. 3 is a schematic view showing the structure of a centrifugal separation detecting device provided with a hole and/or a transparent member in Embodiment 2 of the present invention.
  • Figure 4 is a schematic view showing the structure of a solid phase film supporting film-like member.
  • FIG. 5 is a schematic view showing the structure of the fixture of the solid phase film of FIG. 4.
  • Fig. 6 is a structural schematic view showing the rotary moving connecting device of the solid phase film holder of Fig. 5.
  • Fig. 7 is a schematic view showing the centrifugal rotor of Fig. 1 adopting an outwardly inclined structure.
  • Figure 8 is a schematic view showing the connection structure of the solid phase membrane and the sample tube.
  • Figure 9 is a schematic view showing the structure of a centrifugal rotor solid phase film rotary moving connection device.
  • Fig. 10 is a schematic view showing the structure of a solid-phase film holder with a transparent upper and lower embedding member.
  • Anti-human myoglobin polyclonal antibody (Genagates, USA, catalog number GP301042), anti-human myoglobin monoclonal antibody (Genagates, USA, article number GP300616), spectrophotometer (Shanghai Hanhua Technology Instrument Co., Ltd., 752 UV-Vis spectrophotometer), human myoglobin (Sigma-Aldrich product, catalog number F3879-1G), BioFlow film printer (IMAGENE, USA), Index slitter (A-point, USA), DBF -900 sealing machine (Wenzhou Jiangnan Packing Factory), ACBO dehumidifier (Jiangsu Wuxi Aobo Dehumidifier Company), desktop centrifuge (Eppendoff Company, USA), bovine serum albumin (abbreviated as BSA, SIGMA product, item number: B8894), nitrocellulose membrane (AE 99, supplied by Gengates, USA), polyester cellulose membrane (Reemay 2033, product of Alstrom, USA
  • Preparation of human myoglobin solution Take a known concentration of human myoglobin solution and dilute the configuration 3.125, 6.25, 12.5 with sample dilution buffer (1% BSA, 100 mM glycine, 50 mM PBS, 150 mM NaCl, pH 7.4). 25, 50, 100 ng / ml series of human myoglobin solution.
  • sample dilution buffer 1% BSA, 100 mM glycine, 50 mM PBS, 150 mM NaCl, pH 7.4
  • Preparation of colloidal gold-labeled anti-human myoglobin monoclonal antibody take 10ml of purified water, heat and stir, add 500 ⁇ l of 10% chloroauric acid solution when the water is boiling, heat and boil for 5 minutes, add 500 ⁇ l of 12% trisodium citrate solution. The solution was kept stirring and boiled for 10 minutes, and naturally cooled to room temperature, that is, a colloidal gold solution.
  • Preparation of colloidal gold-labeled adsorption membrane preparation of polyester cellulose membrane pretreatment liquid containing 0.5% PVA (ie, polyvinyl alcohol), 50 mM PBS solution, 0.5% BSA, 0.88% NaCl, pH 7.4, and treated for polymerization
  • PVA polyvinyl alcohol
  • BSA polyvinyl alcohol
  • the ester cellulose film was immersed in the pretreatment liquid for 1 hour at room temperature, and the film was taken out, dried at 37 ° C, sealed for use, or directly used as a dispersion film.
  • the colloidal gold-labeled antibody solution was diluted with colloidal buffer (1% BSA, 3% sucrose, 50 mM PBS, pH 7.4) to an OD530 of 30, the membrane printer was started, the antibody was loaded, the pressurized nitrogen was turned on, and the polyester was taken.
  • the cellulose film was started to print the film, and the film conditions were set as follows: the moving speed of the airbrush was 30 mm/sec, and the liquid pushing speed was 3.0 ⁇ l/cm.
  • the printed film was placed in a dry box and dried at 37 ° C for 6 hours. Store in a sealed bag containing desiccant.
  • the colloidal gold-labeled adsorption film and dispersion film are also the liquid adsorption dispersion devices of the present invention.
  • Polyclonal antibody imprinting Take anti-human myoglobin polyclonal antibody solution and dilute to a concentration of 1 mg/ml with 50 mM phosphate buffer (pH 7.4). Start the film printer, load the antibody, take the PVC sheet with the nitrocellulose membrane (ie, the polyvinyl chloride sheet), start printing the film, and set the film conditions as: the speed of the airbrush 30 mm / sec, liquid propulsion speed 0.5 ⁇ l / cm. The printed film was placed in a 37 ° C dry box and dried for 6 hours, and then the film was placed in a desiccant-containing dry container for storage.
  • Semi-finished product assembly method start the dehumidifier to reduce the humidity in the operating room to less than 25%, paste the absorbent paper film pad and the colloidal gold-labeled adsorption film on both ends of the polyclonal antibody printing film, and then seal the surface with the dry tape. Place the attached test piece on the slitter and cut into 3.5mm test strips. Put the paper strip into the aluminum pouch sealed bag with desiccant, seal it on the sealing machine, and label it.
  • test strip prepared above was taken with the side of the colloidal gold-labeled adsorption film facing up, placed in a centrifuge rotor (outwardly inclined type, diameter 30 mm), and the different concentrations of the formulated gold were added to the colloidal gold-labeled adsorption film.
  • 80 ⁇ l of human myoglobin solution allowed to stand for 1-15 minutes, 2000 rpm/separation of the heart for 1 minute, and then add 80 ul of 50 mM PBS buffer containing 0.05% Tween-20 to the colloidal gold labeled adsorption membrane.
  • test strips 2000 rpm / separation heart 1 minute cleaning, take out the test strip, placed on a colloidal gold quantitative chromatography analyzer (ie detector) to read the digital image of the polyclonal antibody strip, and perform image processing to obtain the corresponding chromaticity value .
  • the control test strips were not subjected to centrifugation, and after standing for the same standing time as described above, they were allowed to stand for another 2.5 minutes, and then the corresponding chromaticity values were read.
  • the detection result of the detection technology using colloidal gold as an indicator shows that the correlation coefficient r detected by the technique of the present invention is 0.9884, and the correlation coefficient r of the prior art detection (not performing centrifugation) is 0.957, P ⁇ 0.05, which is remarkable.
  • the detection results of the prior art show that the technique of the present invention improves the accuracy of the prior art detection.
  • the experimental results are shown in Table 1.
  • Example 2 Comparison of the minimum detection amount of the centrifugal separation detection method of the present invention and the current detection technology
  • the measurement result of colloidal gold as an indicator is analyzed, and the data is statistically processed according to the requirement that the correlation coefficient r value of the related product development is greater than 0.98, and the minimum value when the r value is greater than 0.98 is determined as the minimum detection amount.
  • the pre-station time was 1-15 minutes, and the minimum detection amount in the prior art was 25 or >25 ng/ml.
  • the minimum detection amount using the present invention was 3.125 ng/ml, and the detection sensitivity was Significantly higher than the prior art, the technique of the present invention improves the detection sensitivity of the prior art detection.
  • Table 2 The experimental results are shown in Table 2.
  • Example 3 the effect of the centrifugal separation detection method of the present invention on the detection specificity
  • the samples used for specific detection were A: 50 ng/ml myoglobin, B: 10 ng/ml troponin I, C: 30 ng/ml creatine kinase isoenzyme, D: 80 mg/ml human serum albumin, E: 20 mg/ml cholesterol.
  • the invention uses the colloidal gold as an indicator to detect the above specific detection sample, and the experimental results are shown in Table 3.
  • the average values of the repeated detection of the myoglobin sample by the prior art and the present invention are 50.3 and 51.0 ng/ml, respectively.
  • the detection value of the sample containing myoglobin was below the lower limit of the detection sensitivity of the detection method, and both were negative, and there was no obvious color reaction.
  • Horseradish peroxidase-labeled anti-human myoglobin monoclonal antibody (Genagates, USA, article number GP300616), magnetic particles (MP-COOH-20020, Zhengzhou Inno Bio), pico luminescent reagent (Thermo scientific), chemiluminescence detection (Promega, Glomax Multi JR Detection System), other materials are the same as in Example 1.
  • magnetic particles were labeled with a 1 mg/ml anti-human myoglobin polyclonal antibody by conventional labeling method.
  • the ratio of anti-human myoglobin polyclonal antibody to magnetic particles (w/w) was 3: 1.
  • Three parallel tubes were taken for each concentration, and 100 ⁇ l of magnetic particles labeled with anti-human myoglobin polyclonal antibody were added to each tube, and 100 ⁇ l of each concentration of human myoglobin solution was added to each concentration, and the binding reaction was shaken at 37 ° C.
  • the magnetic particles were separated by magnetic separation, the supernatant was discarded, washed twice with 200 ⁇ l of PBS, the magnetic particles were separated by magnetic separation, the supernatant was discarded, and horseradish peroxidase-labeled anti-human myoglobin was added. 200 ⁇ l of monoclonal antibody, the binding reaction was incubated at 37 ° C for 60 minutes, and the magnetic particles were separated by magnetic separation. The supernatant was discarded, washed twice with 200 ⁇ l of PBS, and the magnetic particles were separated by magnetic separation, and the supernatant was discarded. The magnetic particles were placed in a luminescent cup, and a chemiluminescence detector was placed. 100 ⁇ l of the luminescent substrate working solution was added. When the reaction was carried out for 2 minutes, the luminescence amount was recorded for 6 seconds.
  • the polyester polyester film was pretreated as in Example 1, dried at 37 ° C, and sealed for use.
  • the polyclonal antibody print was used in the same manner as in Example 1 and stored in a desiccant-containing dry container.
  • the side of the chemiluminescence-labeled adsorption film is placed upward, placed in a centrifuge rotor (30 mm in diameter), and different concentrations of human myoglobin are added to the chemiluminescent label adsorption film.
  • 80 ul of solution allowed to stand for 2 minutes, 2000 rpm / separation of the heart for 1 minute, and then add 0.05 ul of 0.05% Tween-20 in PBS buffer to the chemiluminescent label adsorption membrane, 2000 rpm / separate heart for 1 minute
  • 100 ⁇ l of the luminescent substrate working solution was added dropwise to the chemiluminescent labeling adsorption film, and 800 rpm was separated for 30 seconds.
  • the nitrocellulose membrane was peeled off from the PVC substrate, and a chemiluminescence detector was placed, and the amount of luminescence was recorded for 6 seconds.
  • the invention adopts the chemiluminescence detection technology and the current chemiluminescence detection technology to detect the human myoglobin solution, and the experimental results are shown in Table 4. As can be seen from Table 4, both of them exhibit a good linear relationship of concentration, and the correlation coefficient r value They are 0.993 and 0.992 respectively. Description of the present invention with current chemiluminescence technology Similar detection results, but significantly shortened the detection time.
  • Example 5 comparison of the detection results of the centrifugal separation detection method of the present invention and the current chemiluminescence detection technology
  • Standard curve was prepared: a known concentration of human myoglobin solution 3.125, 6.25, 12.5, 25, 50, 100 ng/ml human myoglobin solution was used to detect and draw a standard curve using the present invention and current chemiluminescence detection techniques, respectively. A known concentration of human myoglobin 10 ng/ml was used as a sample to be examined. The other method is the same as in the fourth embodiment.
  • the specific results of the three repeated experiments are shown in Table 5.
  • the measurement results of the current chemiluminescence detection technology show that the content of human myoglobin in the sample to be tested is 9.52 ng/ml, and the measurement result of the present invention shows that the content of human myoglobin in the sample to be tested is 9.82 ng/ml, and the results obtained by the two experimental methods are basically Consistent, there was no statistical difference (P>0.05), but the completion time of the present invention was significantly shorter than the current technology.
  • Fluorescent microspheres fluorescein used as bismuth compound, article number JY-SJ126, Shanghai Jieyi Bio Company
  • EDC Pulierce product, article number 22980
  • NHS Pulierce product, article number 24500
  • fluorescence quantitative analyzer Shanghai scar tissue Company, HG-98
  • Fluorescent microsphere labeling Take 0.5ml fluorescent microspheres, centrifuge 4 times with 0.1M PB of PH7.2, centrifuge at 13000 rpm, reconstitute to 1ml with 0.1M PB at pH 7.2, add 150ug anti-human myoglobin monoclonal The antibody was mixed, 0.1 M PB of pH 7.2 was added to 1.5 ml, 250 ul of 40 mg/ml EDC aqueous solution was added, 250 ul of 40 mg/ml aqueous NHS solution was added, and the mixture was mixed and reacted at room temperature for 60 minutes. 20 mg of BSA was added, mixed, and reacted at room temperature for 60 minutes. The supernatant was aspirated by centrifugation, washed 4 times with 0.05 M Tris pH 7.6, reconstituted to 10 ml with 1% BSA, 0.05 M Tris pH 7.6, and stored at 4 °C.
  • Polyester cellulose membrane pretreatment liquid was the same as in Example 1.
  • the fluorescent microsphere-labeled antibody solution was diluted 3 times with 1% BSA, 0.05 M Tris pH 7.6 buffer, the membrane printer was started, the antibody was loaded, the pressurized nitrogen gas was turned on, the polyester cellulose membrane was taken, and the printing film was started.
  • the film conditions were set as follows: the moving speed of the airbrush was 30 mm/sec, the liquid pushing speed was 5.0 ⁇ l/cm, and the printed film was placed in a dry box, dried at 37 ° C for 6 hours, and then placed in a desiccant-containing seal. Stored in a bag.
  • Polyclonal antibody print preparation same as in Example 1.
  • test strip prepared above was placed with the fluorescently labeled antibody adsorption membrane facing up, placed in a centrifuge rotor (30 mm in diameter), and different concentrations of human myoglobin were added to the fluorescently labeled antibody adsorption membrane. 80 ul of solution, allowed to stand for 2 minutes, 2000 rpm / separation of the heart for 1 minute, and then add 80 ul of PBS buffer containing 0.05% Tween-20 at pH 7.4 to the fluorescently labeled antibody adsorption membrane, 2000 rpm/separate the heart for 1 minute. Wash, remove the test strip, and read the fluorescence value of the polyclonal antibody blot on the fluorescence analyzer.
  • the current technical control test strips were not centrifuged, and after standing for a set period of 2 minutes, they were allowed to stand for another 2.5 minutes, and then the fluorescence value was read.
  • the specific results are shown in Table 6.
  • the technique of the present invention is as described above, and the linear reaction is good, and the correlation coefficient r is 0.995.
  • the linearity is not good, and the sample below 12.5 ng/ml has a luminescence amount close to the background level, and the correlation coefficient r is 0.937.
  • the current detection reaction time of the test strip should be 15 minutes. After the experiment is sampled and allowed to stand for 4.5 minutes, the detection reaction has not been completed, and therefore, the linearity is not good.
  • the present invention significantly shortens the detection time.
  • a goat anti-mouse IgG polyclonal antibody (available from Gengates, Inc., Cat. No. GP301231), the same as in Example 6.
  • Fluorescent microsphere labeling same as in Example 6.
  • Polyclonal antibody imprinting Take anti-human myoglobin polyclonal antibody solution and dilute to a concentration of 1 mg/ml with 50 mM phosphate buffer (pH 7.4). Take goat anti-mouse IgG polyclonal antibody solution with 50mM Phosphate buffer (pH 7.4) was diluted to a concentration of 1 mg/ml.
  • the film printer load the antibody, take the PVC sheet with the nitrocellulose membrane, start printing the film, and print the anti-human myoglobin polyclonal antibody on the same nitrocellulose membrane as the detection line T, goat anti-mouse IgG polyclonal
  • the antibody was used as the quality control line C, and the film conditions were set as follows: the moving speed of the airbrush was 30 mm/sec, the liquid pushing speed was 0.5 ⁇ l/cm, and the printed film was placed in a 37 ° C drying oven, dried for 6 hours, and then The membrane is stored in a dry container containing a desiccant.
  • Semi-finished product assembly method start the dehumidifier to reduce the humidity in the operating room to less than 25%, paste the fluorescent-labeled antibody adsorption film on the detection line of the polyclonal antibody printing film, paste the absorbent paper film pad on the quality control line end, and then seal it with the adhesive tape. Paste the surface. Place the attached test piece on the slitter and cut into 3.5mm test strips. Put the paper strip into the aluminum pouch sealed bag with desiccant, seal it on the sealing machine, and label it.
  • test strip prepared above was placed with the fluorescently labeled antibody adsorption membrane facing up, placed in a centrifuge rotor (30 mm in diameter), and different concentrations of human myoglobin were added to the fluorescently labeled antibody adsorption membrane.
  • the solution and the sample to be tested were each 80 ul, allowed to stand for 2 minutes, 2000 rpm/separation of the heart for 1 minute, and then add PBS buffer containing 0.05% Tween-20 at pH 7.4 to the fluorescently labeled antibody adsorption membrane at 80 ul, 2000 rpm.
  • the current technical control test strip is not centrifuged, and after standing for a set period of time, it is allowed to stand for another 2.5 minutes, then the fluorescence value is read, and the T/C ratio is calculated, a standard curve is drawn, and the myoglobin of the sample to be tested is calculated. concentration.
  • the technique of the present invention is operated as above, the standard curve is linear, and the correlation coefficient r is 0.995, and then the sample is measured.
  • the average of three experiments is 10.84 ng/ml, and the detection error is within 10%, which meets the requirements.
  • the standard curve was linear and the correlation coefficient r was 0.989. Then the sample was measured according to the conditions of 15 minutes. The three average values were 9.49 ng/ml.
  • the detection error is within 10%, which meets the requirements.
  • the specific results of the three repeated experiments are shown in Table 7. Compared with the prior art, the present invention significantly shortens the detection time.
  • Example 8 comparison of the detection results of the centrifugal separation detection method (detector using fluorescence detection) and the current enzyme-linked immunoassay technology
  • Fluorescent microspheres (all fluorescein is bismuth compound, item number JY-SJ126, Shanghai Jieyi Bio Company), EDC (Pierce product, article number 22980), NHS (Pierce product, item number 24500), fluorescence quantitative analyzer (Shanghai ⁇ Bio Company) , HG-98), horseradish peroxidase-labeled anti-human myoglobin monoclonal antibody (Genagates, USA, article number GP300616), o-phenylenediamine, enzyme-linked immunosorbent assay (Bio-Rad, Model 550), Healthy human serum (donated by healthy volunteers), goat anti-mouse IgG polyclonal antibody (available from Gengates, USA, article number GP301231). Others are the same as in the first embodiment.
  • Preparation of human myoglobin solution A known concentration of human myoglobin solution was taken, and a series of human myoglobin solutions of 3.125, 6.25, 12.5, 25, 50, 100 ng/ml were placed in a PBS solution to prepare a standard curve. A known concentration of 8.2 ng/ml human myoglobin healthy human serum was used as a sample to be tested.
  • the experiment uses the fluorescence detection of the present invention and the current enzyme-linked immunosorbent assay to detect the human myoglobin solution and draw a standard curve, and then take the sample to be tested for determination, and calculate the concentration of myoglobin in the sample to be tested by using a standard curve. Make 3 parallel tubes for each sample.
  • the OD490 absorbance value was read on the enzyme-linked immunosorbent assay, and the concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 ng/ml were corresponding.
  • Fluorescent microsphere labeling same as in Example 6.
  • Polyclonal antibody print preparation same as in Example 7.
  • test strip prepared above was placed with the fluorescently labeled antibody adsorption membrane facing up, placed in a centrifuge rotor (30 mm in diameter), and different concentrations of human myoglobin were added to the fluorescently labeled antibody adsorption membrane.
  • the solution and the sample to be tested were each 80 ul, allowed to stand for 2 minutes, 2000 rpm/separation of the heart for 1 minute, and then add PBS buffer containing 0.05% Tween-20 at pH 7.4 to the fluorescently labeled antibody adsorption membrane at 80 ul, 2000 rpm.
  • colloidal gold is used as an indicator, and different concentrations of human myoglobin samples are detected at different centrifugal speeds.
  • the experimental results are shown in Table 9. It can be seen from Table 9 that the detection accuracy is related to the centrifugal speed.
  • the centrifugal speeds of 500, 1000, 2000 rpm are obtained to meet the required test results, and the correlation coefficient r values are all greater than 0.98.
  • Centrifugal speeds of 3000, 4000, 5000 rpm
  • the obtained test results, the correlation coefficient r values are all below 0.98, and the relevant test requirements are not met. It is indicated that the optimal centrifugation speed for detecting myoglobin of the present invention should be below 2000 rpm.
  • Example 10 the influence of the injection mode of the centrifugal separation detection method of the present invention on the detection result
  • a 30 mm diameter circular plate was made of stainless steel plate and punched in the center.
  • the stainless steel circular plate is horizontally fixed to the small DC motor shaft, and the peristaltic pump is mounted to the center of the stainless steel circular plate. Connect the DC motor and the peristaltic pump to the battery.
  • Sample to be tested and cleaning solution The container is fixed above the peristaltic pump.
  • the prepared test strip was pasted with a water-absorbent film pad, and the colloidal gold-labeled adsorbing film was adhered inward to the stainless steel circular plate.
  • One end of the peristaltic pump pipette is placed in the sample to be tested and the cleaning liquid container, and the other end is fixed to the colloidal gold-labeled adsorption film.
  • a three-way switch that changes the direction of the liquid suction is installed on the liquid inlet side of the peristaltic pump pipette. During the experiment, the three-way switch is placed on the side of the sample to be tested, and 40 ul of the sample to be tested is dropped onto the colloidal gold-labeled adsorption film, the centrifuge is turned on, the peristaltic pump is turned on, and the colloidal gold-labeled adsorption film is loaded at a rate of 20 ul/min.
  • the control test strip of the invention adopts the injection method of adding the sample gun and centrifuging on the centrifuge, and after the first sample is applied for 80 ul, it is allowed to stand for 1 minute, then 1000 rpm/separation of the heart for 1 minute, and the cleaning liquid is added 80 ul, 1000 rpm. / Separate the heart for 1 minute, then read the image chromaticity value.
  • Example 11 the influence of the detection method of the centrifugal separation detection method of the present invention on the detection result
  • Fluorescent microsphere labeling same as in Example 6.
  • the test strip prepared above was placed with the fluorescently labeled antibody adsorption membrane facing up, placed in a centrifuge rotor (30 mm in diameter), and different concentrations of human myoglobin were added to the fluorescently labeled antibody adsorption membrane.
  • the solution and the sample to be tested were each 80 ul, allowed to stand for 2 minutes, 2000 rpm/separation of the heart for 1 minute, and then add PBS buffer containing 0.05% Tween-20 at pH 7.4 to the fluorescently labeled antibody adsorption membrane at 80 ul, 2000 rpm. / Separate the heart for 1 minute, remove the test strip, and read the fluorescence value of the detection line on the polyclonal antibody printed film on a fluorescence quantitative analyzer.
  • Bilateral detection is performed in the fluorescence quantitative analysis.
  • Another fluorescent detection probe is installed on the opposite side of the existing fluorescence detection probe.
  • the PVC negative film is torn off during the detection. Other conditions are unchanged, and the detection line on the polyclonal antibody printed film is read. Fluorescence value.
  • the double-side detection of the invention can significantly improve the amount of fluorescence read, and can improve the detection sensitivity.
  • Example 12 the influence of the cleaning step of the centrifugal separation detecting method of the present invention on the detection result
  • the experimental washing step was allowed to stand without washing. Others are the same as in the first embodiment.
  • the centrifugal separation detecting apparatus of the present invention comprises: a sample injection unit 1, a solid phase membrane 2, a centrifugal device 3, and a detector 4.
  • the centrifugal device 3 includes a centrifugal rotor 9, a drive motor 10, and a support base 11 supporting the centrifugal rotor 9, and the drive motor 10 drives the centrifugal rotor 9 to rotate.
  • the centrifugal device In order to protect the centrifugal device, it can be placed in a housing 5.
  • the sample introduction unit 1 includes a liquid phase storage device 6, a sample pump 7, and a sample introduction tube 8.
  • the liquid phase storage device 6 is in communication with the sample introduction tube 8, is disposed in the middle of the centrifugal rotor 9, and is driven into the sample introduction tube 8 by the sample pump 7; the sample introduction tube 8 is disposed on the centrifugal rotor 9.
  • both the centrifuge device and the injection member are connected to a component having a program control speed.
  • the solid phase film 2 and the centrifugal rotor 9 are of a detachable structure; the solid phase film 2 is placed in a solid phase film placing device 26 provided at the outer edge of the centrifugal rotor 9, a solid phase film 2 is provided at the proximal end of the centrifugal rotor 9 with a liquid adsorption dispersing member 15 communicating therewith, and the solid phase membrane 2 is provided at the telecentric end of the centrifugal rotor with a liquid collecting member 16 communicating therewith, and the liquid adsorbing and dispersing member 15 It is in communication with the sample tube 8.
  • the material of the solid phase film 2 is any one of a nitrocellulose membrane, a polyvinylidene fluoride membrane, a nylon membrane and a DEAE cellulose membrane, and the solid phase membrane 2 is provided with a backing on one or both sides;
  • the liquid adsorption dispersion member 15 includes at least one of a colloidal gold-labeled adsorption film, a fluorescently labeled antibody adsorption film, a chemiluminescent label adsorption film, and a dispersion film; and the liquid collection device 16 is made of a water-absorbent material such as absorbent paper and/or water-absorbent gel. Also, a liquid collection container can be used.
  • a solid phase film fixing device for fixing the solid phase film 2 supports the backsheet 17, and the supporting film 17 can be selected from a PVC plate, a transparent plastic plate, a plexiglass plate or the like.
  • the solid phase film fixing device of Fig. 5 is a lateral flow test strip buckle clamping member 20, specifically including a hole member 18, a spotting groove 19, an observation window 21, and a liquid collecting member outlet 22.
  • the corresponding portion of the sampling tank 19 is a liquid adsorption dispersion member, and the corresponding portion of the observation window 21 is a solid phase membrane 2, and the liquid collection member outlet 22 corresponds to The location is a liquid collection component 16 (either a water absorbent material or a liquid collection container).
  • the solid phase film placing device 26 employs a rotary movable fixing device including a columnar projection 23, a cylindrical projection 23 and a hole shape provided in the lateral flow test strip buckle clamping member 20.
  • Component 18 is matched.
  • the columnar projection 23 is a columnar structure provided on the centrifugal rotor 9.
  • the columnar projection 23 on the centrifugal rotor 9 is inserted into the hole-like member 18 of the lateral flow test strip buckle clamping member 20, and one end of the injection tube 8 is connected to the spotting groove 19, and the other end is liquid.
  • the phase reservoir 6 is connected and the liquid phase is driven into the sample tube 8 via the injection pump and then to the spotting tank 19.
  • a columnar projection 23 for fixing the solid phase film holder e.g., the lateral flow test strip buckle clamping member 20
  • a columnar projection 23 for fixing the solid phase film holder e.g., the lateral flow test strip buckle clamping member 20
  • the hole member 18 is evenly distributed on the centri
  • the detector 4 is provided on the outer edge of the centrifugal rotor 9, and is provided on one side of the solid phase film 2.
  • the solid phase membrane 2 and the sample tube 8 are connected to each other, and the sample tube 8 is placed above the solid phase membrane 2, directly connected to the liquid adsorption dispersion member 15 or contacted by a liquid phase 25 drop contact. .
  • the liquid phase 25 is directly loaded or dropped through the sample introduction tube 8 to the liquid adsorption dispersion member 15.
  • another solid phase film fixing device provided for fixing the solid phase film 2 specifically includes a hard transparent lower cover sheet 27, a hard transparent upper cover sheet 28, a front bare empty interlayer 29, and a rear bare space. Interlayer 30.
  • the liquid phase is centrifuged, passes through the front bare space interlayer 29, enters the liquid adsorption dispersion member 15, flows through the solid phase film 2, and the liquid phase after the reaction is discharged from the rear bare space interlayer 30.
  • the center inclined type centrifugal separation detecting device and the horizontal type centrifugal separating device of the present invention have the same types of components, including the injection part 1, the solid phase film 2, the centrifugal device 3, and the detection. 4 and the corresponding composition.
  • the difference is that the centrifugal rotor 9 is inclined outwardly with the center outwardly inclined centrifugal rotor 9A as a center, and the solid phase film is outwardly inclined by the lateral flow test strip buckle clamping member 20 on the center outwardly inclined centrifugal rotor 9A. Placed, it can be placed in the outer surface of the center outwardly inclined centrifugal rotor 9A or in a centrifugal hole or interlayer provided inside.
  • Embodiment 15 Preparation of a centrifugal separation detecting device provided with a hole and/or a transparent member of the present invention
  • FIG. 3 it is the same as that in Embodiment 13, except that a flat intermediate portion of the centrifugal rotor 9 is provided with a hole and/or a transparent member 12, and the hole and/or the transparent member 12 makes the solid phase film 2 Directly opposite the upper detector 13 and the lower detector 14 of the detector, that is, the aperture and/or the transparent member 12 exposes the solid phase film 2 between the upper detector 13 and the lower detector 14 of the detector for upper detection.
  • the lower detector 14 and the lower detector 14 can simultaneously read the detection data of the solid phase film 2.
  • Planetary geared motor ie drive motor of centrifugal device, output speed 500-5000 rpm, power 60w, custom
  • potentiometer thickness 1.5mm iron plate
  • micro peristaltic pump ie injection pump in injection part, Baoding Chuangrui pump industry, model BW100
  • myoglobin colloidal gold detection card ie integrated structure of solid phase membrane solid phase membrane and solid phase membrane holder, Changzhou Bowendi company
  • human myoglobin Samples were taken, Sigma-Aldrich product catalog number F3879-1G), colloidal gold quantitative chromatography analyzer (ie detector, Norwegian Skannex product).
  • Preparation of human myoglobin solution take a known concentration of human myoglobin solution, dilute the configuration with sample dilution buffer (1% BSA, 100 mM glycine, 50 mM PBS, 150 mM NaCl, pH 7.4) 25, 50, 100, 300, 500 ng / ml series of human myoglobin solution.
  • sample dilution buffer 1% BSA, 100 mM glycine, 50 mM PBS, 150 mM NaCl, pH 7.4
  • the myoglobin colloidal gold detection card is outwardly coated with a water-absorbent film, and the colloidal gold-labeled adsorption film (ie, the liquid adsorption-dispersing member) is attached to the outer side of the iron plate in the inward direction.
  • the colloidal gold-labeled adsorption film ie, the liquid adsorption-dispersing member
  • One end of the peristaltic pump pipette is placed on the sample to be tested and cleared
  • the other end of the washing liquid container is fixed to the test card spotting tank (colloidal gold mark adsorption film).
  • a three-way switch that changes the direction of the liquid suction is installed on the liquid inlet side of the peristaltic pump pipette.
  • a colloidal gold quantitative chromatography analyzer was attached to the pre-cut iron plate.
  • the myoglobin colloidal gold detection card is attached to the side of the centrifuge rotor with the proximal end of the colloidal gold-labeled adsorption membrane, and one end of the peristaltic pump pipette is placed in the sample to be tested and the cleaning liquid container, and the other end is fixed.
  • To the test card spotting tank (colloidal gold mark adsorption film). Open the peristaltic pump pipette tee to the sample to be inspected. Turn on the peristaltic pump and adjust the speed to 50 ul/min. See that the sample liquid to be tested flows out from the sampling tank end of the peristaltic pump pipette and flows onto the colloidal gold-labeled adsorption film.
  • Another muscle myoglobin colloidal gold test card add 100ul of a series of human myoglobin solution of known concentration to each card, let stand for 20 minutes, and place the test card on the colloidal gold quantitative chromatograph to read the result. . The experiment was repeated three times and the results were averaged.
  • the existing colloidal gold solid phase membrane detection product is used, and the planar centrifugal separation apparatus of the present invention is compared with the conventional conventional method, and the results are as shown in Table 1.
  • the technique of the present invention and the prior art detection and sample true value are observed. Correlation.
  • the results show that the correlation coefficient r detected by the technique of the present invention is 0.998, and the correlation coefficient r detected by the prior art is 0.983, P ⁇ 0.05, which is significantly better than the detection result of the prior art, and the detection time of the prior art detection is 20 minutes.
  • the technology of the present invention is 3.5 minutes, indicating that the technology of the present invention not only improves the accuracy of the prior art detection, but also shortens the detection time.
  • Small tabletop centrifuge ie centrifugal device, Eppendorf, Minispin, angular rotor, output speed 1000-10000 rpm
  • 1.5mm thick iron plate 8mm diameter iron rod
  • micro peristaltic pump ie injection pump for injection parts, Baoding Chuangrui Pump Industry, model BW100
  • myoglobin colloidal gold detection card ie integrated structure of solid phase membrane solid phase membrane and solid phase membrane holder, Changzhou Bowendi company
  • human myoglobin ie Samples to be tested, Sigma-Aldrich product, catalog number F3879-1G
  • colloidal gold quantitative chromatography analyzer ie detector, Norwegian Skannex product.
  • the cutting iron rod is 50mm long, and the circular iron plate with a diameter of 150mm is cut.
  • One end of the iron rod is fixed at the center of the circular iron plate, and the other end of the iron rod is vertically fixed with the shaft of the centrifugal rotor of the small centrifuge.
  • a micro-peristaltic pump, battery, and liquid phase container are placed over the plane of the iron bar.
  • the myoglobin colloidal gold detection card is placed in the angular rotor centrifugal hole with the water absorption membrane pad outward and the colloidal gold labeled adsorption film in the inward direction.
  • One end of the peristaltic pump pipette is placed in the sample to be tested and the cleaning liquid container, and the other end is fixed to the test card spotting tank (colloidal gold-labeled adsorption film).
  • a three-way switch that changes the direction of the liquid suction is installed on the liquid inlet side of the peristaltic pump pipette.
  • the myoglobin colloidal gold detection card is placed on the side of the colloidal gold-labeled adsorption membrane (ie, the liquid adsorption dispersion member) as a proximal end in the angular rotor centrifugal hole, and one end of the peristaltic pump pipette is placed in the sample to be tested and The other end of the cleaning solution container is fixed to the test card sampling tank (colloidal gold-labeled adsorption film). Open the peristaltic pump pipette tee to the sample to be inspected. Turn on the peristaltic pump and adjust the speed to 50 ul/min.
  • the sample liquid to be tested flows out from the sampling tank end of the peristaltic pump pipette and flows onto the colloidal gold-labeled adsorption film.
  • Turn on the small centrifuge adjust the speed to 1000 rpm, and after centrifugation for 2 minutes, turn the three-way to the cleaning solution, adjust the speed of the small centrifuge to 2000 rpm, adjust the speed of the peristaltic pump to 150 ul/min, and close after centrifugation for 1 minute.
  • Peristaltic pump adjust the speed of the small centrifuge to 5000 rpm, continue centrifugation for 30 seconds, and close the small centrifuge. Remove the test card and place it on a colloidal gold quantitative chromatograph to read the results.
  • a series of human myoglobin solutions at concentrations of 25, 50, 100, 300, 500 ng/ml were sequentially tested. The experiment was repeated three times and the results were averaged.
  • Another muscle myoglobin colloidal gold test card add 100ul of a series of human myoglobin solution of known concentration to each card, let stand for 20 minutes, and place the test card on the colloidal gold quantitative chromatograph to read the result. . The experiment was repeated three times and the results were averaged.
  • the existing colloidal gold solid phase membrane detection product is used, and the external oblique centrifugal separation device of the present invention is compared with the existing conventional method, and the experimental results are shown in Table 2.
  • the technology of the present invention and the prior art detection and sample are observed. The relevance of the value.
  • the results show that the correlation coefficient r detected by the technique of the present invention is 0.996, and the correlation coefficient r detected by the prior art is 0.987, P ⁇ 0.05, which is significantly better than the detection results of the prior art, and the detection time of the prior art detection is 20 minutes.
  • the technology of the present invention is 3.5 minutes, indicating that the technology of the present invention not only improves the accuracy of the prior art detection, but also shortens the detection time.
  • Planetary geared motor ie the drive motor of the centrifugal device, output speed 500-5000 rpm, power 60w, custom), potentiometer, thickness 1.5mm iron plate, micro peristaltic pump (ie injection pump for injection parts, Baoding Sharp pump industry, model BW100), myoglobin colloidal gold detection card (ie integrated structure of solid phase membrane solid phase membrane and solid phase membrane holder, Changzhou Bowendi company), diameter 3mm iron rod (ie rotary movement) Fixation device columnar protrusion), human myoglobin (ie sample to be tested, Sigma-Aldrich product, catalog number F3879-1G), colloidal gold quantitative chromatography analyzer (ie detector, Norwegian Skannex product).
  • micro peristaltic pump ie injection pump for injection parts, Baoding Sharp pump industry, model BW100
  • myoglobin colloidal gold detection card ie integrated structure of solid phase membrane solid phase membrane and solid phase membrane holder, Changzhou Bowendi company
  • diameter 3mm iron rod ie
  • Another muscle myoglobin colloidal gold test card add 100ul of a series of human myoglobin solution of known concentration to each card, let stand for 20 minutes, and place the test card on the colloidal gold quantitative chromatograph to read the result. . The experiment was repeated three times and the results were averaged.
  • the existing colloidal gold solid phase film detecting product is used, and the planar centrifugal separating device with the rotary mobile fixing device of the present invention is compared with the existing conventional method, and the experimental results are shown in Table 3, and the technical and technical aspects of the present invention are observed.
  • the prior art detects the correlation with the true value of the sample.
  • the results show that the correlation coefficient r detected by the technique of the present invention is 0.998, and the correlation coefficient r detected by the prior art is 0.988, P ⁇ 0.05, which is significantly better than the detection results of the prior art, and the detection time of the prior art detection is 20 minutes.
  • the technology of the present invention is 3.5 minutes, indicating that the technology of the present invention not only improves the accuracy of the prior art detection, but also shortens the detection time.
  • the present invention uses a centrifugal device to drive the liquid phase to be flowed and cleaned on the solid phase membrane, thereby improving the capture and binding ability of the analyte, reducing the background noise interference of the solid phase membrane, improving the detection sensitivity of the method, and realizing the present High sensitivity detection with detection reagents.
  • the present invention uses a centrifugal device to drive the liquid phase detected on the solid phase membrane, which changes the existing membrane detection technology by relying on natural flow and the liquid is reduced as the flow on the membrane is prolonged, and the liquid can be kept in the membrane.
  • the uniform flow on the surface ensures the uniformity of the binding of the analyte on the membrane, which can improve the inspection. Measure accuracy.
  • the invention uses a centrifugal device to drive the detection liquid phase to flow on the solid phase membrane, keeps the liquid flowing uniformly on the membrane, shortens the detection time, and has the advantages of rapid detection.
  • the centrifugal device drives the liquid flow and the injection pump to be injected, and the operation steps are simple, and it is convenient to develop a more convenient miniaturized detection device.
  • multi-step and multi-step drive control is adopted, which involves the defect of transposition and movement of the detection sample, the detection phase and the reaction carrier.
  • the operation steps of the invention are simple and easy to realize automatic operation.
  • the method of the invention has the characteristics of high sensitivity, full quantification and automation, and has the detection technology of rapid detection and simple use of equipment; not only is convenient to use, waste of raw materials is reduced, but also work efficiency is obviously improved, and is applied to detection, analysis and separation. Many areas.

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Abstract

一种离心分离检测方法及装置。该方法包括如下步骤：采用离心装置（3）驱动液相流经固相膜（2），液相包括含有待检物的待检样品和检测相；液相流经固相膜（2）时，与固相膜（2）上包被的待检物特异性结合物结合形成检测指示剂-待检物-待检物特异性结合物的复合物，复合物被捕获固定到固相膜（2）上，检测器（4）通过检测被间接固定到固相膜上的检测指示剂的量，以检测待检物的含量；该装置包括进样部件（1）、固相膜（2）、离心装置（3）和检测器（4）；进样部件（1）设于离心转子（9）的中部，并与离心转子（9）相连接；固相膜（2）设于离心转子（9）上，与进样管（8）相连接；检测器（4）设于固相膜（2）的一侧或两侧。具有灵敏度高、检测时间短的特点。

Description

一种离心分离检测方法及装置 技术领域
本发明涉及一种离心分离检测方法及装置，具体涉及一种离心分离检测方法和一种离心分离检测装置，分别属于免疫检测技术和仪器分析领域。
背景技术
免疫学检测技术是应用免疫学原理设计的测定抗原、抗体、免疫细胞及化学成分等的实验手段，广泛用于来源于人体和动物体可进行疾病诊断和健康检测的样品以及用于环境、药物分析、食品和工业分析的样品。常用的有免疫浊度技术、固相酶免疫测定技术、化学发光检测技术、免疫荧光标记技术、流式细胞术、胶体金技术等。
免疫浊度技术，也称免疫浊度法是可溶性抗原、抗体在液相中特异结合，产生一定大小的复合物，形成光的折射或吸收，测定这种折射或吸收后的透射光或散射光作为计算单位，用于定量检测，但检测灵敏度低，不适合于微量检测。固相酶免疫测定技术基于抗原或抗体的固相化及抗原或抗体的酶标记，结合在固相载体表面的抗原或抗体保持其免疫学活性，抗原或抗体的酶结合物既保留其免疫学活性，又保留酶的活性，在测定时，把受检标本(测定其中的抗体或抗原)和酶标抗原或抗体按不同的步骤与固相载体表面的抗原或抗体起反应，具有灵敏度高、线性响应范围宽和易于实现自动化等显著优点，但检测反应时间长限制了其使用。免疫化学发光检测技术是一种高灵敏的微量及痕量分析技术，具有操作方便、灵敏度高、线性响应范围宽和易于实现自动化等显著优点，广泛地应用于环境、临床、药物分析、食品和工业分析中，也是基于抗原或抗体的固相分离手段及抗原或抗体的发光试剂标记技术，但检测反应时间长及对检测设备的要求高也影响其使用。免疫荧光标记技术、流式细胞术、胶体金技术也是常用的检测技术被广泛使用，但均有其相应的不足，检测反应时间长或灵敏度、准确性欠缺是普遍存在的不足。
高灵敏度、快速、小型化、全定量、自动化是目前临床免疫检测技术产品的发展趋势，但现有的都无法同时实现上述功能。因此，开发一种能够实 现既具有高灵敏度、全定量、自动化特点，同时又具有检测快速、设备简单的新的检测设备，不仅使用方便、减少浪费，同时也可显著提高工作效率，在检测和分析、分离的诸多领域具有重要的实用意义。
发明公开
本发明的目的是提供一种离心分离检测方法，本发明的另一个目的是提供一种离心分离检测装置，本发明检测方法具有灵敏度高、检测时间短的特点，本发明离心分离检测装置具有检测速度快、高灵敏度、使用方便的特点。
本发明提供的离心分离检测方法，包括如下步骤：采用离心装置驱动液相流经固相膜，所述液相包括含有待检物的待检样品和检测相，所述检测相为含有能够与所述待检物直接或间接形成特异性结合的检测指示剂的物质；所述液相流经固相膜时，与所述固相膜上包被的待检物特异性结合物结合形成检测指示剂-待检物-待检物特异性结合物的复合物，所述复合物被捕获固定到所述固相膜上，检测器通过检测被间接固定到所述固相膜上的检测指示剂的量，即检测到所述待检物的含量。
上述的方法中，所述方法采用的装置中，所述离心装置的旋转部件采用平面型或由中心向外倾斜型。
上述的方法中，所述方法采用的装置中，所述离心装置的旋转部件平面的中部设有：所述待检样品和所述检测相各自的储存装置，所述待检样品和所述检测相的进样装置及驱动装置；
所述离心装置的旋转部件平面的外沿设有所述固相膜的放置装置。
本发明中，所述固相膜的进样近端与液体吸附分散装置连接，进样远端与液体吸附装置连接；
所述液体吸附分散装置采用具有快速的液体分散特性的固相材料，所述固相材料为多聚酯纤维素膜和/或玻璃纤维素膜，具体可为胶体金标记吸附膜、荧光标记抗体吸附膜、化学发光标记吸附膜和/或分散膜；所述液体吸附装置采用吸水性材料，所述吸水性材料为吸水纸和/或吸水凝胶。
上述的方法中，所述方法采用的装置中，所述检测器设于所述固相膜的一侧或两侧。
上述的方法中，所述离心装置的转速和所述进样装置的进样速度均采用程序控制方式；
所述离心装置的转速为200～10000r/min，具体可为500～5000r/min、800～3000r/min或800～2000r/min；
经所述离心装置离心的时间可为1～5min，具体可为1min或1～3min。
上述的方法中，所述固相膜为硝酸纤维素膜、聚偏氟乙烯膜、尼龙膜、DEAE纤维素膜，且所述固相膜的一面或两面带有背衬；
所述检测器包括吸光度、荧光、化学发光和图像颜色数字处理的检测器中的任一种。
本发明中，所述聚偏氟乙烯膜简称PVDF膜；
所述DEAE纤维素膜指的是将二乙氨乙基(DEAE)引入纤维素分子后制成的纸状薄膜，是一种弱碱型阴离子交换材料。
上述的方法中，所述待检物为具有免疫活性的蛋白质或与蛋白质偶联产生免疫活性的物质；
所述待检物特异性结合物为与所述待检物特异性结合的抗原或抗体；
所述检测指示剂为胶体金属、染料、荧光素和化学发光物质中的至少一种。
上述的方法中，所述胶体金属为胶体金、胶体硒和胶体金磁微粒中的至少一种；
所述荧光素为异硫氰酸荧光素(简称FITC)、四乙基罗丹明、四甲基异硫氰酸罗丹明、藻红蛋白(简称PE)、多甲藻黄素叶绿素蛋白(简称PerCP)、碘化丙啶(简称PI)、别藻青蛋白(简称APC)和铕化合物中的至少一种，其中铕化合物具体可为氧化铕；
所述化学发光物质为鲁米诺和异鲁米诺及其衍生物类、吖啶酯及吖淀酰胺类、(金钢烷)-1,2-二氧乙烷及其衍生物和三联吡啶钌中的至少一种。
上述的方法中，所述复合物形成有如下任一种：
1)所述待检物特异性结合物包含有同时与所述待检物和所述检测相形成特异性结合的一级中间相；
2)所述待检物特异性结合物包含有同时与所述一级中间相和所述检测相形成特异性结合的二级中间相；
所述一级中间相和所述二级中间相均为具有特异性结合能力的抗原、抗体、亲和素、生物素及其类似物中的至少一种；
所述方法中还包括在所述结合反应之后用清洗相清洗所述固相膜的步骤，所述清洗相为磷酸盐缓冲液、碳酸盐缓冲液和Tris缓冲液中的至少一种。
本发明离心分离检测方法应用于免疫产品的含量检测中。
本发明提供的离心分离检测装置，它包括进样部件、固相膜、离心装置和检测器；
所述离心装置包括由驱动电机驱动的离心转子和支撑底座，所述离心转子以所述支撑底座为支撑；
所述进样部件设于所述离心转子的中部，并与离心转子相连接；所述进样部件包括液相储存装置和进样管；所述液相储存装置与所述进样管相连通；
所述固相膜设于所述离心转子上，与所述进样管相连接；
所述检测器设于所述固相膜的一侧或两侧。
上述的离心分离检测装置中，所述固相膜置于固相膜放置装置中，且所述固相膜放置装置设于所述离心转子上；
所述固相膜与所述离心转子为可拆装式结构。
上述的离心分离检测装置中，所述固相膜放置装置为旋转移动式固定装置和/或槽形挤压式固定装置
上述的离心分离检测装置中，所述固相膜通过固相膜固定器固定，所述固相膜固定器选自固相膜支撑底片样部件、侧向流试纸条扣卡样部件和透明质上下包埋式部件中至少一种。
上述的离心分离检测装置中，所述透明质上下包埋式部件为所述固相膜的上下两侧由硬质透明材料覆盖，所述固相膜对应侧的所述硬质透明材料的面积大于或等于所述固相膜的面积。
上述的离心分离检测装置中，所述固相膜在与所述进样管相连的一端还设有与之相连通的液体吸附分散部件，所述固相膜于所述离心转子的远心端设有与之相连通的液体收集部件，所述液体吸附分散部件与所述进样管相连通。
上述的离心分离检测装置中，所述进样部件包含有进样泵，所述进样泵驱动所述液相储存装置中的液体进入所述进样管。
上述的离心分离检测装置中，所述液相储存装置包括待检样品储存装置和检测相储存装置；
所述待检样品储存装置和所述检测相储存装置均与所述进样管相连通，且均由所述进样泵驱动。
上述的离心分离检测装置中，所述液相储存装置还包括清洗液储存装置，所述清洗液储存装置与所述进样管相连通，且由所述进样泵驱动。
上述的离心分离检测装置中，所述离心转子采用平面型或由中心向外倾斜型；
所述离心装置设有一外壳。
上述的离心分离检测装置中，所述旋转移动式固定装置为在所述离心转子上设置的柱状凸起的连接装置，所述固相膜固定器上设有与所述柱状凸起的连接装置相匹配的孔状部件。
上述的离心分离检测装置中，所述离心装置、所述进样部件和所述检测器均设有程序控制装置。
上述的离心分离检测装置中，所述进样管和所述固相膜采用可拆装式相连接；
所述液体吸附分散部件与所述进样管采用可拆装式相连通。
上述的离心分离检测装置中，所述固相膜的材料采用硝酸纤维素膜、聚偏氟乙烯膜、尼龙膜和DEAE纤维素膜中的任一种，且所述固相膜的一面或两面设有背衬；
所述液体吸附分散部件包括胶体金标记吸附膜、荧光标记抗体吸附膜、化学发光标记吸附膜、多聚酯纤维分散膜和玻璃纤维分散膜中的至少一种；
所述检测器包括吸光度、荧光、化学发光和图像数字处理的检测器中的任一种。
本发明中，所述液体收集装置可采用吸水性材料制成，具体为吸水纸和/或吸水凝胶，所述液体收集装置可为液体收集容器。
上述的离心分离检测装置中，所述离心转子的平面中间段设有孔和/或设有透明部件，且所述孔和/或所述透明部件均使所述固相膜直接暴露于所 述检测器。
本发明所述的离心分离检测装置在免疫产品检测中的应用。
上述离心分离检测装置的应用中，所述免疫产品包括抗原、抗体、免疫细胞和化学成分中至少一种。
本发明由于采取以上技术方案，其具有以下优点：
1、本发明采用离心装置驱动检测的液相在固相膜上流动以及清洗，提高了待检物的捕获结合能力，降低了固相膜的背景噪声干扰，提高方法检测灵敏度，实现了以现有检测试剂的高灵敏度检测。
2、本发明采用离心装置驱动检测的液相在固相膜上流动，改变了现有的膜检测技术依靠自然流动且液体随着在膜上流程的延长而降低的现状，能够保持液体在膜上匀速流动，保证了待检物在膜上结合的均一性，可提高检测准确性。
3、本发明采用离心装置驱动检测液相在固相膜上流动，保持了液体在膜上匀速流动，缩短了检测时间，具有快速检测的优点。克服了现有膜检测技术依靠自然流动、液体在膜上的流动速度随着时间而减慢的问题，完成一项检测一般需要15分钟以上的时间的不足。
4、本发明的以离心装置驱动液体流动和进样泵进样，操作步骤简单，便于开发更为便捷的小型化检测设备。克服了现有的高灵敏度检测技术中均采用多步骤、多环节驱动控制，涉及检测样本、检测相以及反应载体的换位和移动的不足。
5、本发明操作步骤简单，易于实现自动化操作。本发明方法具有高灵敏度、全定量、自动化的特点，同时又具有检测快速、使用设备简单的检测技术；不仅使用方便、减少原料的浪费，同时也显著提高工作效率，应用于检测和分析、分离的诸多领域。
附图说明
图1为本发明实施例1中离心分离检测装置的示意图。
图2为图1增加部件的示意图。
图3为本发明实施例2中设有孔和/或透明部件离心分离检测装置结构的示意图。
图4为固相膜支撑底片样部件结构示意图。
图5为图4中固相膜的固定器结构示意图。
图6为图5中固相膜固定器旋转移动式连接装置结构示意图。
图7为图1中离心转子采用向外倾斜型结构的示意图。
图8为固相膜与进样管连接结构示意图。
图9为离心转子固相膜旋转移动式连接装置结构示意图。
图10为固相膜固定器透明质上下包埋式部件的结构示意图。
图中标记如下：
1进样部件；2固相膜；3离心装置；4检测器；5外壳；6液相储存装置；7进样泵；8进样管；9离心转子；9A中心向外倾斜型离心转子；10驱动电机；11支撑底座；12孔和/或透明部件；13上检测器；14下检测器；15液体吸附分散部件；16液体收集部件；17固相膜支撑底片；18孔状部件；19点样槽；20侧向流试纸条扣卡样部件；21观察窗；22液体收集部件出口；23柱状凸起；24进样管固定器；25液相；26固相膜放置装置；27硬质透明下盖片；28硬质透明上盖片；29前裸空夹层；30后裸空夹层。
实施发明的最佳方式
下述实施例中的实验方法，如无特别说明，均为常规方法。
下面结合附图对本发明进行进一步说明，但本发明并不局限于以下实施例。
实施例1、本发明离心分离检测方法与现行检测技术的检测准确性的比较
一、实验材料
抗人肌红蛋白多克隆抗体(美国Genagates公司，其产品目录号为GP301042)，抗人肌红蛋白单克隆抗体(美国Genagates公司，货号GP300616)，分光光度计(上海箐华科技仪器有限公司，752紫外可见分光光度计)，人肌红蛋白(Sigma-Aldrich产品，产品目录号为F3879-1G)，BioFlow印膜仪(美国IMAGENE公司)，Index切条机(美国A-point公司)，DBF-900封口机(温州江南包装厂)，ACBO除湿机(江苏无锡奥波除湿机公司)，台式离心机(美国Eppendoff公司)，牛血清白蛋白(简称BSA， SIGMA产品，货号：B8894)，硝酸纤维素膜片(AE 99，由美国Genagates公司提供)，多聚酯纤维素膜(Reemay 2033，美国Alstrom公司产品)，吸水纸膜垫(Grade 470，美国S&S公司产品)，氯金酸(SIGMA产品，货号：B8894)，胶体金定量层析分析仪(挪威Skannex产品)。
二、实验方法
人肌红蛋白溶液的配制：取已知浓度的人肌红蛋白溶液，用样品稀释缓冲液(1％BSA，100mM甘氨酸，50mM PBS,150mM NaCl，pH7.4)稀释配置3.125、6.25、12.5、25、50、100ng/ml的系列人肌红蛋白溶液。
胶体金标记抗人肌红蛋白单克隆抗体的制备：取10ml纯净水，加热搅拌，待水沸腾时加入500μl 10％氯金酸溶液，加热煮沸5分钟，加入500μl 12％柠檬酸三钠溶液，保持此溶液搅拌沸腾10分钟，自然降温至室温，即胶体金溶液。取胶体金溶液体积10ml，用10％碳酸钾调pH至8.3，迅速加入抗人肌红蛋白单克隆抗体100μg，至10μg/ml终浓度，晃动烧杯混匀，室温放置30分钟，迅速加入10％牛血清白蛋白溶液100ul，使终浓度为1％，同时摇动烧杯，室温放置30分钟，12000rpm离心20分钟，小心吸出上清液；再加入5ml 50mM磷酸盐(PBS)缓冲液，pH7.4，将沉淀悬浮，12000rpm离心20分钟，吸出上清液，将沉淀溶于1.0ml含有1％的牛血清白蛋白和3％蔗糖的磷酸缓冲液内，4℃避光保存。
胶体金标记吸附膜制备：配制含有0.5％PVA(即聚乙烯醇)、50mM PBS液、0.5％BSA、0.88％NaCl，pH 7.4的多聚酯纤维素膜预处理液，置待处理的多聚酯纤维素膜于预处理液内，室温浸泡1小时，将膜取出，置37℃干燥后密封备用，也可直接作为分散膜使用。取胶体金标记抗体溶液，用胶体缓冲液(1％BSA，3％蔗糖，50mM PBS,pH7.4)稀释至OD530为30，启动印膜仪，装载抗体，开启加压氮气，取多聚酯纤维素膜，开始印膜，设定印膜条件为：喷笔移动速度30mm/秒，液体推进速度3.0μl/cm，将印制后的膜放入干燥箱内，37℃干燥6小时，然后置于含干燥剂的密封袋内保存使用。胶体金标记吸附膜和分散膜同时也是本发明所述的液体吸附分散装置。
多克隆抗体印膜制备：取抗人肌红蛋白多克隆抗体溶液，用50mM磷酸盐缓冲液(pH 7.4)稀释至1mg/ml浓度。启动印膜仪，装载抗体，取贴有硝酸纤维素膜的PVC片(即聚氯乙烯片)，开始印膜，设定印膜条件为：喷笔移动速度 30mm/秒，液体推进速度0.5μl/cm。将印制好的膜放入37℃干燥箱内，干燥6小时，然后将膜置于含干燥剂的干燥容器内保存使用。
半成品组装方法：启动除湿机使操作室内的湿度降低至25％以下，在多克隆抗体印膜两端分别粘贴吸水纸膜垫及胶体金标记吸附膜，然后用不干胶带封贴表面。置粘贴好的检测片于切条机上，切成3.5mm试纸条。将纸条放入有干燥剂的铝珀密封袋内，在封口机上封口，加贴标签。
取上述制备的试纸条，以胶体金标记吸附膜的一侧朝上，置于离心机转子(外向倾斜型，直径为30mm)内，向胶体金标记吸附膜上滴加配制的不同浓度的人肌红蛋白溶液80ul，静置1-15分钟，2000转/分离心1分钟，再向胶体金标记吸附膜上滴加pH7.4的含0.05％吐温-20的50mM PBS缓冲液80ul，2000转/分离心1分钟清洗，取出试纸条，放置于胶体金定量层析分析仪(即检测器)上读取多克隆抗体印迹条带的数字图像，进行图像处理获得相应的色度数值。对照试纸条不做离心处理，在设定的上述相同的静置时间后，再静置2.5分钟，然后读取相应的色度数值。
实验重复三次，结果取平均值。统计学计算不同预静置时间试纸条的检测值。
三、实验结果
本发明以胶体金为指示剂的检测技术测定结果显示本发明技术检测的相关系数r平均值为0.9884，现有技术检测(不做离心处理)的相关系数r为0.957，P＜0.05，显著优于现有技术的检测结果，说明本发明技术提高了现有技术检测的准确性。实验结果如表1所示。
表1本发明以胶体金为指示剂的检测结果准确性分析(单位：信号值)
Figure PCTCN2016086842-appb-000001
实施例2、本发明离心分离检测方法与现行检测技术的最低检出量的比较
一、实验材料
同实施例1
二、实验方法
同实施例1
三、实验结果
本发明以胶体金为指示剂的测定结果，分析采用相关产品开发常用的相关系数r值大于0.98的要求对数据进行了统计处理，以r值大于0.98时的最小值定为最低检出量。结果同样的实验条件下，预静置时间1-15分钟，采用现有技术的最低检出量为25或＞25ng/ml，采用本发明的最低检出量均为3.125ng/ml，检测灵敏度显著高于现有技术，说明本发明技术提高了现有技术检测的检测灵敏度。实验结果如表2所示。
表2本发明以胶体金为指示剂的检测灵敏度分析(单位：信号值)
Figure PCTCN2016086842-appb-000002
实施例3、本发明离心分离检测方法对检测特异性的影响
一、实验材料
同实施例1
二、实验方法
同实施例1
特异性检测所用样品为A：50ng/ml肌红蛋白、B：10ng/ml肌钙蛋白I、 C：30ng/ml肌酸激酶同工酶、D：80mg/ml人血清白蛋白、E：20mg/ml胆固醇。
三、实验结果
本发明以胶体金为指示剂的检测上述特异性检测样品，实验结果如表3所示，现行技术和本发明技术对肌红蛋白样品重复检测平均值分别为50.3和51.0ng/ml，其它不含有肌红蛋白的样品的检测值在检测方法的检测灵敏度下限以下，均为阴性，且无明显的显色反应。
表3本发明与现行技术检测肌红蛋白特异性的结果比较(单位：ng/ml)
Figure PCTCN2016086842-appb-000003
实施例4、本发明离心分离检测方法与现行化学发光检测技术的检测性能的比较
一、实验材料
辣根过氧化物酶标记抗人肌红蛋白单克隆抗体(美国Genagates公司，货号GP300616)、磁微粒(MP-COOH-20020，郑州英诺生物)、pico发光试剂(Thermo scientific)、化学发光检测仪(Promega,Glomax Multi JR Detection System)，其它材料同实施例1。
二、实验方法
人肌红蛋白溶液的配制：同实施例1。
1、现行化学发光检测技术组
磁微粒的标记，采用常规标记方法，用1mg/ml抗人肌红蛋白多克隆抗体对磁微粒进行标记，抗人肌红蛋白多克隆抗体和磁微粒的用量比(w/w)为3:1。各浓度检测取三个平行管，每管加入用抗人肌红蛋白多克隆抗体标记的磁微粒100μl，再分别加入浓度为对应浓度的人肌红蛋白溶液各100μl，结合反应在37℃震摇温育60分钟，用磁分离器吸附分离磁微粒，弃上清，加入PBS 200μl清洗三次，用磁分离器吸附分离磁微粒，弃上清，加入辣根过氧化物酶标记抗人肌红蛋白单克隆抗体200μl，结合反应在37℃震摇温育60分钟，用磁分离器吸附分离磁微粒，弃上清，加入PBS 200μl清洗三次，用磁分离器吸附分离磁微粒，弃上清，转移磁微粒至发光杯，置化学发光检测仪，加入100μl发光底物工作液，反应进行2分钟时，记录发光量6秒钟。
2、本发明组
多聚酯纤维素膜预处理同实施例1，置37℃干燥后密封备用。
多克隆抗体印膜同实施例1，置于含干燥剂的干燥容器内保存使用。
化学发光标记吸附膜的制备：取预处理的多聚酯纤维素膜，用50mM PBS缓冲液(pH 7.4)稀释辣根过氧化物酶标记抗人肌红蛋白单克隆抗体0.15mg/ml，启动印膜仪，装载抗体，开启加压氮气，取多聚酯纤维素膜，开始印膜，设定印膜条件为：喷笔移动速度30mm/秒，液体推进速度3.0μl/cm，将印制后的膜冷冻干燥，置4℃密封保存备用。
半成品组装方法：同实施例1。
取上述制备的试纸条，以化学发光标记吸附膜的一侧朝上，置于离心机转子(直径为30mm)内，向化学发光标记吸附膜上滴加配制的不同浓度的人肌红蛋白溶液80ul，静置2分钟，2000转/分离心1分钟，再向化学发光标记吸附膜上滴加pH7.4的含0.05％吐温-20的PBS缓冲液80ul，2000转/分离心1分钟清洗，向化学发光标记吸附膜滴加100μl发光底物工作液，800转/分离心30秒，从PVC底片上剥取硝酸纤维素膜，置化学发光检测仪，记录发光量6秒钟。
三、实验结果
采用本发明以化学发光检测技术和现行化学发光检测技术对人肌红蛋白溶液进行检测，实验结果如表4所示，由表4可知，两者均呈现良好的浓度线性关系，相关系数r值分别为0.993和0.992。说明本发明具有与现行化学发光技 术相似的检测效果，但显著缩短了检测时间。
表4本发明与现行化学发光检测技术的检测性能的发光量比较
Figure PCTCN2016086842-appb-000004
实施例5、本发明离心分离检测方法与现行化学发光检测技术检测结果的比较
一、实验材料
同实施例4。
二、实验方法
制作标准曲线：取已知浓度的人肌红蛋白溶液3.125、6.25、12.5、25、50、100ng/ml人肌红蛋白溶液，分别采用本发明和现行化学发光检测技术检测并绘制标准曲线。以已知浓度的人肌红蛋白10ng/ml作为待检样本。其它方法同实施例4。
三、实验结果
三次重复实验的具体结果如表5所示。现行化学发光检测技术测定结果显示待检样本人肌红蛋白的含量为9.52ng/ml，本发明测定结果显示待检样本人肌红蛋白的含量为9.82ng/ml，两种实验方法所得结果基本一致，无统计学差异(P>0.05)，但本发明的完成实验时间明显短于现行技术。
表5本发明与现行化学发光检测技术检测结果的比较(单位：ng/ml)
Figure PCTCN2016086842-appb-000005
实施例6、本发明离心分离检测方法与现行荧光免疫检测技术检测性能的比较
一、实验材料
荧光微球(所用荧光素为铕化合物，货号JY-SJ126，上海杰一生物公司)、EDC(Pierce产品，货号22980)、NHS(Pierce产品，货号24500)、、荧光定量分析仪(上海巾帼生物公司，HG-98)、其它同实施例1。
二、实验方法
人肌红蛋白溶液的配制：同实施例1。
荧光微球标记：取0.5ml荧光微球，使用PH7.2的0.1M PB离心清洗4次，13000rpm离心，用pH7.2的0.1M PB复溶至1ml，加入150ug抗人肌红蛋白单克隆抗体，混匀，加入pH7.2的0.1M PB至1.5ml，加入250ul的40mg/ml的EDC水溶液，加入250ul 40mg/ml的NHS水溶液，混匀，室温反应60分钟。加入20mg的BSA，混匀，室温反应60分钟。离心吸弃上清，使用0.05M Tris pH7.6离心清洗4次后，用1％BSA、0.05M Tris pH7.6复溶至10ml待用，4℃保存。
荧光标记抗体吸附膜的制备：多聚酯纤维素膜预处理液同实施例1。取荧光微球标记抗体溶液，用1％BSA、0.05M Tris pH7.6缓冲液稀释3倍，启动印膜仪，装载抗体，开启加压氮气，取多聚酯纤维素膜，开始印膜，设定印膜条件为：喷笔移动速度30mm/秒，液体推进速度5.0μl/cm，将印制后的膜，放入干燥箱内，37℃干燥6小时，然后置于含干燥剂的密封袋内保存使用。
多克隆抗体印膜制备：同实施例1。
半成品组装方法：同实施例1。
取上述制备的试纸条，以荧光标记抗体吸附膜的一侧朝上，置于离心机转子(直径为30mm)内，向荧光标记抗体吸附膜上滴加配制的不同浓度的人肌红蛋白溶液80ul，静置2分钟，2000转/分离心1分钟，再向荧光标记抗体吸附膜上滴加pH7.4的含0.05％吐温-20的PBS缓冲液80ul，2000转/分离心1分钟清洗，取出试纸条，在荧光定量分析仪上读取多克隆抗体印迹条带的荧光值。
现行技术对照试纸条不做离心处理，在设定的2分钟静置时间后，再静置2.5分钟，然后读取荧光值。
实验重复三次，结果取平均值。
三、实验结果
具体结果如表6所示。本发明技术经如上操作，线性反应良好，相关系数r值为0.995。采用现有技术测定，点样后静置2分，再静置2.5分，线性不佳，12.5ng/ml以下的样品，其发光量接近本底水平，相关系数r值为0.937。本试纸条的现行检测反应时间应该为15分钟，本实验点样后静置4.5分钟，检测反应尚未完成，因此，线性不佳。与现有技术比较，本发明显著缩短了检测时间。
表6本发明与现行免疫荧光检测技术的检测性能的发光量比较
Figure PCTCN2016086842-appb-000006
实施例7、本发明离心分离检测方法与现行免疫荧光检测技术检测结果的比较
一、实验材料
羊抗鼠IgG多克隆抗体(美国Genagates公司提供，货号GP301231)，其它同实施例6。
二、实验方法
人肌红蛋白溶液的配制：同实施例1。配制10ng/ml已知浓度的人肌红蛋白待检样品。
荧光微球标记：同实施例6。
荧光微球膜的印制：同实施例6。
荧光标记抗体吸附膜的制备：同实施例6。
多克隆抗体印膜制备：取抗人肌红蛋白多克隆抗体溶液，用50mM磷酸盐缓冲液(pH 7.4)稀释至1mg/ml浓度。取羊抗鼠IgG多克隆抗体溶液，用50mM 磷酸缓冲液(pH 7.4)稀释至1mg/ml浓度。启动印膜仪，装载抗体，取贴有硝酸纤维素膜的PVC片，开始印膜，同一硝酸纤维素膜上印制抗人肌红蛋白多克隆抗体作为检测线T、羊抗鼠IgG多克隆抗体作为质控线C，设定印膜条件为：喷笔移动速度30mm/秒，液体推进速度0.5μl/cm，将印制好的膜放入37℃干燥箱内，干燥6小时，然后将膜置于含干燥剂的干燥容器内保存使用。
半成品组装方法：启动除湿机使操作室内的湿度降低至25％以下，在多克隆抗体印膜检测线端粘贴荧光标记抗体吸附膜，质控线端粘贴吸水纸膜垫，然后用不干胶带封贴表面。置粘贴好的检测片于切条机上，切成3.5mm试纸条。将纸条放入有干燥剂的铝珀密封袋内，在封口机上封口，加贴标签。
取上述制备的试纸条，以荧光标记抗体吸附膜的一侧朝上，置于离心机转子(直径为30mm)内，向荧光标记抗体吸附膜上滴加配制的不同浓度的人肌红蛋白溶液及待测样品各80ul，静置2分钟，2000转/分离心1分钟，再向荧光标记抗体吸附膜上滴加pH7.4的含0.05％吐温-20的PBS缓冲液80ul，2000转/分离心1分钟清洗，取出试纸条，在荧光定量分析仪上读取多克隆抗体印制膜上检测线T和质控线C的荧光值，并计算T/C比值，绘制标准曲线，计算待检样品肌红蛋白浓度。
现行技术对照试纸条不做离心处理，在设定的静置时间后，再静置2.5分钟，然后读取荧光值，并计算T/C比值，绘制标准曲线，计算待检样品肌红蛋白浓度。
实验重复三次，结果取平均值。
三、实验结果
本发明技术经如上操作，标准曲线线性良好，相关系数r值为0.995，随即进行了样品测定，三次实验平均10.84ng/ml，检测误差在10％以内，符合要求。采用现有技术测定，点样后静置2分，再静置2.5分，检测标准曲线线性不佳，相关系数r值为0.937。随后将总的点样后静置时间延长至现行产品检测的15分钟，标准曲线线性良好，相关系数r值为0.989，随即按照15分钟的条件进行了样品测定，三次平均值为9.49ng/ml，检测误差在10％以内，符合要求。三次重复实验的具体结果如表7所示。与现有技术比较，本发明显著缩短了检测时间。
表7本发明以免疫荧光检测技术的检测结果分析(单位：ng/ml)
Figure PCTCN2016086842-appb-000007
实施例8、本发明离心分离检测方法(检测器采用荧光检测)与现行酶联免疫检测技术检测结果的比较
一、实验材料
荧光微球(所有荧光素为铕化合物，货号JY-SJ126，上海杰一生物公司)、EDC(Pierce产品，货号22980)、NHS(Pierce产品，货号24500)、荧光定量分析仪(上海巾帼生物公司，HG-98)、、辣根过氧化物酶标记抗人肌红蛋白单克隆抗体(美国Genagates公司，货号GP300616)、邻苯二胺、酶联免疫检测仪(Bio-Rad,Model 550)、健康人血清(健康自愿者捐赠)、羊抗鼠IgG多克隆抗体(美国Genagates公司提供，货号GP301231)。其它同实施例1。
二、实验方法
人肌红蛋白溶液的配制：取已知浓度的人肌红蛋白溶液，用PBS溶液配置3.125、6.25、12.5、25、50、100ng/ml的系列人肌红蛋白溶液，用于制作标准曲线。以已知浓度的8.2ng/ml人肌红蛋白健康人血清作为待检样品。
实验采用本发明荧光检测与现行酶联免疫检测技术检测人肌红蛋白溶液并绘制标准曲线，然后取待检样品测定，用标准曲线计算待检样品中肌红蛋白的浓度。每个样品做3个平行管。
1、现行酶联免疫检测技术组
采用96孔酶联板，每管加入抗人肌红蛋白多克隆抗体100μl，4℃过夜包被，清洗三次，再分别加入人肌红蛋白溶液或待检样品100μl，结合反应在37℃温育120分钟，清洗三次，加入抗人肌红蛋白单克隆抗体100μl，结合反应在37℃温育60分钟，清洗三次，弃上清，加入辣根过氧化物酶标记羊抗鼠IgG多克隆抗体100μl，结合反应在37℃温育60分钟，清洗三次，弃上清，加入100μl显色液(配方：0.1M柠檬酸2.43ml，0.2M磷酸氢二钠2.57ml，邻苯二胺5mg，过氧化氢5μl)，避光5分钟，加入2M硫酸终止反应。置酶联免疫检测仪上读取OD490吸光值，3.125、6.25、12.5、25、50、100ng/ml浓度对应 OD值分别为0.182、0.215、0.256、0.398、0.791、1.212，绘制标准曲线，r＝0.993，计算待检样品中人肌红蛋白含量。
2、本发明组
荧光微球标记：同实施例6。
荧光微球膜的印制：同实施例6。
荧光标记抗体吸附膜的制备：同实施例6。
多克隆抗体印膜制备：同实施例7。
半成品组装方法：同实施例7。
取上述制备的试纸条，以荧光标记抗体吸附膜的一侧朝上，置于离心机转子(直径为30mm)内，向荧光标记抗体吸附膜上滴加配制的不同浓度的人肌红蛋白溶液及待测样品各80ul，静置2分钟，2000转/分离心1分钟，再向荧光标记抗体吸附膜上滴加pH7.4的含0.05％吐温-20的PBS缓冲液80ul，2000转/分离心1分钟清洗，取出试纸条，在荧光定量分析仪上读取多克隆抗体印印制膜上检测线T和质控线C的荧光值，并计算T/C比值，0.001、0.005、0.024、0.138、0.373、0.683，r＝0.991，绘制标准曲线，计算待检样品肌红蛋白浓度。
三、实验结果
具体结果如表8所示。现行酶联免疫检测技术测定结果显示待检样本人肌红蛋白的含量为7.89ng/ml，本发明测定结果显示待检样本人肌红蛋白的含量为8.19ng/ml，两种实验方法所得结果基本一致，无统计学差异(P>0.05)，但本发明的完成实验时间明显短于现行技术。
表8本发明荧光检测与现行酶联免疫检测技术检测结果的比较(单位：ng/ml)
Figure PCTCN2016086842-appb-000008
实施例9、本发明离心分离检测方法离心速度对检测结果的影响
一、实验材料
同实施例1。
二、实验方法
同实施例1。
三、实验结果
本发明以胶体金为指示剂，采用不同离心速度，检测了不同浓度的人肌红蛋白样品，实验结果如表9所示。由表9可知，检测准确性与离心速度有关，500、1000、2000转/分的离心速度获得符合要求的检测结果，相关系数r值均大于0.98。3000、4000、5000转/分的离心速度获得的检测结果，其相关系数r值均低于0.98，没有达到相关检测要求。说明本发明进行肌红蛋白检测的最佳离心速度应在2000转/分以下。
表9离心速度对检测结果的影响
Figure PCTCN2016086842-appb-000009
实施例10、本发明离心分离检测方法的进样方式对检测结果的影响
一、实验材料
小型行星减速电机(输出转速1000转/分，功率100w)，不锈钢板，蠕动泵(保定创锐泵业，型号BT100LC)，其它同实施例1。
二、实验方法
将小型行星减速电机直立安装，转轴向上。用不锈钢板制作直径30mm圆形板，中央打孔。将不锈钢圆板水平固定到小型直流电机轴上，将蠕动泵安装到不锈钢圆板的中央。将直流电机和蠕动泵与电池连接。将待测样本和清洗液 容器固定在蠕动泵的上方。将制备的试纸条以吸水膜垫向外，胶体金标记吸附膜向内的方向粘贴至不锈钢圆板上。蠕动泵吸液管一端放置于待测样本和清洗液容器内，另一端固定至胶体金标记吸附膜上。蠕动泵吸液管进液侧安装有可以改变吸液流方向的三通开关。实验时先置三通开关于待测样本一侧，向胶体金标记吸附膜滴加待测样本40ul，开启离心机，开启蠕动泵，以20ul/min速度向胶体金标记吸附膜加样，2分钟后，旋转三通开关至清洗液一侧，蠕动泵速度调至160ul/min，30秒时关闭蠕动泵，继续离心30秒，关闭离心机，取试纸条用胶体金定量层析分析仪测定肌红蛋白条带部位的图像色度值。其它同实施例1。
本发明对照试纸条采用加样枪滴加并在离心机上离心的进样方式，第一次加样80ul后静置1分钟，然后1000转/分离心1分钟，加清洗液80ul，1000转/分离心1分钟，然后读取图像色度值。
三、实验结果
实验结果如表10所示。两实验组结果比较，自动进样方式的检测结果优于手动点样，相关系数r值分别为0.996含0.983。说明自动进样优于手动进样。
表10本发明进样方式对检测结果的影响(结果为图像色度值)
Figure PCTCN2016086842-appb-000010
实施例11、本发明离心分离检测方法的检测方式对检测结果的影响
一、实验材料
同实施例6
二、实验方法
人肌红蛋白溶液的配制：同实施例1。
荧光微球标记：同实施例6。
荧光微球膜的印制：同实施例6。
荧光标记抗体吸附膜的制备：同实施例6。
多克隆抗体印膜制备：同实施例7
半成品组装方法：同实施例7。
取上述制备的试纸条，以荧光标记抗体吸附膜的一侧朝上，置于离心机转子(直径为30mm)内，向荧光标记抗体吸附膜上滴加配制的不同浓度的人肌红蛋白溶液及待测样品各80ul，静置2分钟，2000转/分离心1分钟，再向荧光标记抗体吸附膜上滴加pH7.4的含0.05％吐温-20的PBS缓冲液80ul，2000转/分离心1分钟清洗，取出试纸条，在荧光定量分析仪上读取多克隆抗体印制膜上检测线的荧光值。
双侧检测是在荧光定量分析内，在现有的荧光检测探头相对一侧安装另一个荧光检测探头，检测时撕掉PVC底片，其它条件不变，读取多克隆抗体印制膜上检测线的荧光值。
三、实验结果
具体结果如表11所示。与常规单侧检测比较，本发明采用双侧检测可以明显提高读取的荧光量，可以提高检测灵敏度。
表11本发明检测方式对检测结果发光量的影响
Figure PCTCN2016086842-appb-000011
实施例12、本发明离心分离检测方法清洗步骤对检测结果的影响
一、实验材料
同实施例1
二、实验方法
对照实验清洗步骤静置，不清洗。其它同实施例1。
三、实验结果
实验结果如表12所示。两实验组结果比较，清洗的检测结果优于不清洗，相关系数r值分别为0.996含0.979。说明实验进样后伴有清洗的步骤是必要的。
表12本发明清洗步骤对检测结果的影响(结果为图像色度值)
Figure PCTCN2016086842-appb-000012
实施例13、水平型离心分离检测装置的制备
如图1所示，本发明离心分离检测装置包括：进样部件1、固相膜2、离心装置3和检测器4。
如图2所示，离心装置3包括离心转子9、驱动电机10和支撑离心转子9的支撑底座11，驱动电机10驱动离心转子9转动。为了保护离心装置，可将其设于一外壳5中。
如图2所示，进样部件1包括由液相储存装置6、进样泵7和进样管8。液相储存装置6与进样管8相连通，设于离心转子9的中部，且由进样泵7驱动进入进样管8中；进样管8设于离心转子9上。为了控制离心装置的速度和进样装置的进样速度，离心装置和进样部件均与具有程序控制速度的部件相连接。
如图2和图4所示，固相膜2与离心转子9为可拆装式结构；固相膜2放置于设在离心转子9的外沿的固相膜放置装置26中，固相膜2于离心转子9的近心端设有与之相连通的液体吸附分散部件15，固相膜2于离心转子的远心端设有与之相连通的液体收集部件16，液体吸附分散部件15与进样管8相连通。其中，固相膜2的材料采用硝酸纤维素膜、聚偏氟乙烯膜、尼龙膜和DEAE纤维素膜中的任一种，且固相膜2的一面或两面设有背衬； 液体吸附分散部件15包括胶体金标记吸附膜、荧光标记抗体吸附膜、化学发光标记吸附膜和分散膜中的至少一种；液体收集装置16采用吸水性材料，如吸水纸和/或吸水凝胶，也可用液体收集容器。
如图4、图5所示，为固定固相膜2设置的一种固相膜固定装置，如图4中支撑底片17，支撑底片17可选择PVC板、透明塑料板、有机玻璃板等。图5中固相膜固定装置为侧向流试纸条扣卡样部件20，具体包括孔状部件18、点样槽19、观察窗21和液体收集部件出口22。将固相膜结构放入侧向流试纸条扣卡样结构20后，点样槽19对应部位为液体吸附分散部件，观察窗21的对应部位为固相膜2，液体收集部件出口22对应部位为液体收集部件16(为吸水性材料或液体收集容器)。
如图6、图9所示，固相膜放置装置26采用旋转移动式固定装置，它包括柱状凸起23，柱状凸起23与侧向流试纸条扣卡样部件20上设置的孔状部件18相匹配。柱状凸起23为设置在离心转子9上的柱状结构。使用时将位于离心转子9上的柱状凸起23插入侧向流试纸条扣卡样部件20的孔状部件18内，将进样管8的一端连接至点样槽19，另一端与液相存储器6连接，液相经进样泵驱动进入进样管8，然后加至点样槽19。离心转子9上均匀分布有用于通过孔状部件18固定固相膜固定器(如侧向流试纸条扣卡样部件20)的柱状凸起23。
如图1、图2所示，检测器4设于离心转子9的外沿，其设于固相膜2的一侧。
如图8所示，固相膜2与进样管8连接结构，将进样管8置于固相膜2的上方，与液体吸附分散部件15直接接触连接或通过液相25滴加接触连接。液相25通过进样管8直接加载或滴加到液体吸附分散部件15。
如图10所示，为固定固相膜2设置的另一种固相膜固定装置，具体包括硬质透明下盖片27、硬质透明上盖片28、前裸空夹层29和后裸空夹层30。检测时液相通过离心，经前裸空夹层29，进入液体吸附分散部件15，流经固相膜2，反应后的液相从后裸空夹层30排出。
实施例14、中心向外倾斜型离心分离检测装置的制备
如图7所示，本发明中心向外倾斜型离心分离检测装置与水平型离心分离装置所含部件种类相同，包括进样部件1、固相膜2、离心装置3和检测 器4及相应的组成。区别在于离心转子9采用中心向外倾斜型离心转子9A为中心向外倾斜型，固相膜通过侧向流试纸条扣卡样部件20在中心向外倾斜型离心转子9A上呈外向倾斜型放置，可以放置在中心向外倾斜型离心转子9A的外表面或内部设置的离心孔或夹层内。
实施例15、本发明设有孔和/或透明部件离心分离检测装置的制备
如图3所示，其与实施例13中设置相同，不同的是：将离心转子9的平面中间段设有孔和/或透明部件12，且孔和/或透明部件12使固相膜2直接与检测器的上检测器13、下检测器14相对，即孔和/或透明部件12使固相膜2暴露于检测器的上检测器13、下检测器14之间，以使上检测器13、下检测器14可以同时读取固相膜2的检测数据。
本发明离心分离检测装置的应用实验：
实验一、本发明实施例13中平面型离心分离装置的制备及装置的检测效果：
1、实验材料
行星减速电机(即离心装置的驱动电机，输出转速500-5000转/分，功率60w，定制)，电位器，厚度1.5mm铁板，微型蠕动泵(即进样部件中的进样泵，保定创锐泵业，型号BW100)，肌红蛋白胶体金检测卡(即固相膜的固相膜和固相膜固定器的一体结构，常州博闻迪公司产品)，人肌红蛋白(即待检样品，Sigma-Aldrich产品，产品目录号为F3879-1G)、胶体金定量层析分析仪(即检测器，挪威Skannex产品)。
2、实验方法
1)人肌红蛋白溶液的配制：取已知浓度的人肌红蛋白溶液，用样品稀释缓冲液(1％BSA，100mM甘氨酸，50mM PBS,150mM NaCl，pH7.4)稀释配置25、50、100、300、500ng/ml的系列人肌红蛋白溶液。
2)实验步骤：将行星减速电机直立安装在一预切割的铁板上，转轴向上。用铁板制作直径30mm圆形板，中央打孔。将铁板水平固定到行星减速电机轴上，将蠕动泵安装到铁板的中央。将行星减速电机与电位器相连接，再与电池连接。将蠕动泵与电池连接。将待测样本和清洗液容器固定在铁板上。将肌红蛋白胶体金检测卡以吸水膜垫向外，胶体金标记吸附膜(即液体吸附分散部件)向内的方向粘贴在铁板外侧上。蠕动泵吸液管一端放置于待测样本和清 洗液容器内，另一端固定至检测卡点样槽(胶体金标记吸附膜)上。蠕动泵吸液管进液侧安装有可以改变吸液流方向的三通开关。将胶体金定量层析分析仪固定在预切割的铁板上。
取肌红蛋白胶体金检测卡，以胶体金标记吸附膜的一侧近心端粘贴固定在离心转子平面上，将蠕动泵吸液管一端放置于待测样本和清洗液容器内，另一端固定至检测卡点样槽(胶体金标记吸附膜)上。将蠕动泵吸液管三通开向待检样本。开启蠕动泵，调速至50ul/min，见待检样本液自蠕动泵吸液管点样槽端流出，流至胶体金标记吸附膜上。开启行星减速电机，用电位器调速至1000转/分，离心2分钟后，将三通转向清洗液，调行星减速电机转速至2000转/分，蠕动泵速度调至150ul/min，离心1分钟后关闭蠕动泵，调行星减速电机转速至5000转/分，继续离心30秒，关闭行星减速电机。取下检测卡，放置于胶体金定量层析分析仪上读取结果。依次对浓度为25、50、100、300、500ng/ml的系列人肌红蛋白溶液进行检测。实验重复三次，结果取平均值。
另取肌红蛋白胶体金检测卡，按照说明书每个卡滴加100ul已知浓度的系列人肌红蛋白溶液，静置20分钟，将检测卡放置于胶体金定量层析分析仪上读取结果。实验重复三次，结果取平均值。
3、实验结果
采用现有的胶体金固相膜检测产品，用本发明平面型离心分离装置和现有的常规方法作比较，结果如表1所示，观察本发明技术和现有技术检测与样品真值的相关性。结果显示本发明技术检测的相关系数r为0.998，现有技术检测的相关系数r为0.983，P＜0.05，显著优于现有技术的检测结果，同时，现有技术检测的检测时间为20分钟，本发明技术为3.5分钟，说明本发明技术不仅提高了现有技术检测的准确性，同时还缩短了检测时间。
表1本发明平面型离心分离装置的比对实验(单位：ng/ml)
Figure PCTCN2016086842-appb-000013
实验二、本发明实施例14中外斜型离心分离装置的效果检测
1、实验材料
小型台式离心机(即离心装置，Eppendorf，Minispin，角转子，输出转速1000-10000转/分)，厚度1.5mm铁板，直径8mm铁棒，微型蠕动泵(即进样部件的进样泵，保定创锐泵业，型号BW100)，肌红蛋白胶体金检测卡(即固相膜的固相膜和固相膜固定器的一体结构，常州博闻迪公司产品)，人肌红蛋白(即待检样品，Sigma-Aldrich产品，产品目录号为F3879-1G)、胶体金定量层析分析仪(即检测器，挪威Skannex产品)。
2、实验方法
人肌红蛋白溶液的配制：同实验一中2-1)。
切割铁棒长50mm，切割直径150mm圆形铁板，将铁棒一端固定在圆形铁板中心，铁棒的另一端与小型离心机离心转子中轴竖直固定。在铁棒平面上方固定放置微型蠕动泵、电池和液相容器。将肌红蛋白胶体金检测卡以吸水膜垫向外、胶体金标记吸附膜向内的方向呈外斜型固定放置在角转子离心孔内。蠕动泵吸液管一端放置于待测样本和清洗液容器内，另一端固定至检测卡点样槽(胶体金标记吸附膜)上。蠕动泵吸液管进液侧安装有可以改变吸液流方向的三通开关。
取肌红蛋白胶体金检测卡，以胶体金标记吸附膜(即液体吸附分散部件)的一侧作为近心端放置在角转子离心孔内，将蠕动泵吸液管一端放置于待测样本和清洗液容器内，另一端固定至检测卡点样槽(胶体金标记吸附膜)上。将蠕动泵吸液管三通开向待检样本。开启蠕动泵，调速至50ul/min，见待检样本液自蠕动泵吸液管点样槽端流出，流至胶体金标记吸附膜上。开启小型离心机，调速至1000转/分，离心2分钟后，将三通转向清洗液，调小型离心机转速至2000转/分，蠕动泵速度调至150ul/min，离心1分钟后关闭蠕动泵，调小型离心机转速至5000转/分，继续离心30秒，关闭小型离心机。取出检测卡，放置于胶体金定量层析分析仪上读取结果。依次对浓度为25、50、100、300、500ng/ml的系列人肌红蛋白溶液进行检测。实验重复三次，结果取平均值。
另取肌红蛋白胶体金检测卡，按照说明书每个卡滴加100ul已知浓度的系列人肌红蛋白溶液，静置20分钟，将检测卡放置于胶体金定量层析分析仪上读取结果。实验重复三次，结果取平均值。
3、实验结果
采用现有的胶体金固相膜检测产品，用本发明外斜型离心分离装置和现有的常规方法作比较，实验结果如表2所示，观察本发明技术和现有技术检测与样品真值的相关性。结果显示本发明技术检测的相关系数r为0.996，现有技术检测的相关系数r为0.987，P＜0.05，显著优于现有技术的检测结果，同时，现有技术检测的检测时间为20分钟，本发明技术为3.5分钟，说明本发明技术不仅提高了现有技术检测的准确性，同时还缩短了检测时间。
表2本发明外斜型离心分离装置的比对实验(单位：ng/ml)
Figure PCTCN2016086842-appb-000014
实验三、本发明实施例15中带有旋转移动式固定装置的平面型离心分离装置检测效果
1、实验材料
行星减速电机(即离心装置的驱动电机，输出转速500-5000转/分，功率60w，定制)，电位器，厚度1.5mm铁板，微型蠕动泵(即进样部件的进样泵，保定创锐泵业，型号BW100)，肌红蛋白胶体金检测卡(即固相膜的固相膜和固相膜固定器的一体结构，常州博闻迪公司产品)，直径3mm铁棒(即旋转移动式固定装置柱状凸起)、人肌红蛋白(即待检样品，Sigma-Aldrich产品，产品目录号为F3879-1G)、胶体金定量层析分析仪(即检测器，挪威Skannex产品)。
2、实验方法
人肌红蛋白溶液的配制：同实验一中2-1)。
切割长10mm的直径3mm铁棒5个，竖直焊接到实施例1平面型离心分离装置离心转子的外侧远心平面上。取肌红蛋白胶体金检测卡，在靠近加样槽的近心端打孔，将焊接的竖直铁棒插入肌红蛋白胶体金检测卡近心端孔内，以胶体金标记吸附膜(即液体吸附分散部件)的一侧近心端固定在离心转子平面上，将蠕动泵吸液管一端放置于待测样本和清洗液容器内，另一端固定至检测卡点样槽(胶体金标记吸附膜)上。将蠕动泵吸液管三通开向待检样本。开启蠕动泵，调速至50ul/min，见待检样本液自蠕动泵吸液管点样槽端流出，流 至胶体金标记吸附膜上。开启行星减速电机，用电位器调速至1000转/分，离心2分钟后，将三通转向清洗液，调行星减速电机转速至2000转/分，蠕动泵速度调至150ul/min，离心1分钟后关闭蠕动泵，调行星减速电机转速至5000转/分，继续离心30秒，关闭行星减速电机。取下检测卡，放置于胶体金定量层析分析仪上读取结果。依次对浓度为25、50、100、300、500ng/ml的系列人肌红蛋白溶液进行检测。实验重复三次，结果取平均值。
另取肌红蛋白胶体金检测卡，按照说明书每个卡滴加100ul已知浓度的系列人肌红蛋白溶液，静置20分钟，将检测卡放置于胶体金定量层析分析仪上读取结果。实验重复三次，结果取平均值。
3、实验结果
采用现有的胶体金固相膜检测产品，用本发明带有旋转移动式固定装置的平面型离心分离装置和现有的常规方法作比较，实验结果如表3所示，观察本发明技术和现有技术检测与样品真值的相关性。结果显示本发明技术检测的相关系数r为0.998，现有技术检测的相关系数r为0.988，P＜0.05，显著优于现有技术的检测结果，同时，现有技术检测的检测时间为20分钟，本发明技术为3.5分钟，说明本发明技术不仅提高了现有技术检测的准确性，同时还缩短了检测时间。
表3本发明旋转移动式固定离心分离装置的比对实验(单位：ng/ml)
Figure PCTCN2016086842-appb-000015
工业应用
1、本发明采用离心装置驱动检测的液相在固相膜上流动以及清洗，提高了待检物的捕获结合能力，降低了固相膜的背景噪声干扰，提高方法检测灵敏度，实现了以现有检测试剂的高灵敏度检测。
2、本发明采用离心装置驱动检测的液相在固相膜上流动，改变了现有的膜检测技术依靠自然流动且液体随着在膜上流程的延长而降低的现状，能够保持液体在膜上匀速流动，保证了待检物在膜上结合的均一性，可提高检 测准确性。
3、本发明采用离心装置驱动检测液相在固相膜上流动，保持了液体在膜上匀速流动，缩短了检测时间，具有快速检测的优点。克服了现有膜检测技术依靠自然流动、液体在膜上的流动速度随着时间而减慢的问题，完成一项检测一般需要15分钟以上的时间的不足。
4、本发明的以离心装置驱动液体流动和进样泵进样，操作步骤简单，便于开发更为便捷的小型化检测设备。克服了现有的高灵敏度检测技术中均采用多步骤、多环节驱动控制，涉及检测样本、检测相以及反应载体的换位和移动的不足。
5、本发明操作步骤简单，易于实现自动化操作。本发明方法具有高灵敏度、全定量、自动化的特点，同时又具有检测快速、使用设备简单的检测技术；不仅使用方便、减少原料的浪费，同时也显著提高工作效率，应用于检测和分析、分离的诸多领域。

Claims (27)

  1. 一种离心分离检测方法，包括如下步骤：采用离心装置驱动液相流经固相膜，所述液相包括含有待检物的待检样品和检测相，所述检测相为含有能够与所述待检物直接或间接形成特异性结合的检测指示剂的物质；所述液相流经固相膜时，与所述固相膜上包被的待检物特异性结合物结合形成检测指示剂-待检物-待检物特异性结合物的复合物，所述复合物被捕获固定到所述固相膜上，检测器通过检测被间接固定到所述固相膜上的检测指示剂的量，以检测所述待检物的含量。
  2. 根据权利要求1所述的方法，其特征在于：所述方法采用的装置中，所述离心装置的旋转部件采用平面型或由中心向外倾斜型。
  3. 根据权利要求1或2所述的方法，其特征在于：所述方法采用的装置中，所述离心装置的旋转部件平面的中部设有：所述待检样品和所述检测相各自的储存装置，所述待检样品和所述检测相的进样装置及驱动装置；
    所述离心装置的旋转部件平面的外沿设有所述固相膜的放置装置。
  4. 根据权利要求1所述的方法，其特征在于：所述方法采用的装置中，所述检测器设于所述固相膜的一侧或两侧。
  5. 根据权利要求4所述的方法，其特征在于：所述离心装置的转速和所述进样装置的进样速度均采用程序控制方式；
    所述离心装置的转速为200～10000r/min；
    经所述离心装置离心的时间为1～5min。
  6. 根据权利要求1所述的方法，其特征在于：所述固相膜为硝酸纤维素膜、聚偏氟乙烯膜、尼龙膜、DEAE纤维素膜，且所述固相膜的一面或两面带有背衬；
    所述检测器包括吸光度、荧光、化学发光和图像颜色数字处理的检测器中的任一种。
  7. 根据权利要求1所述的方法，其特征在于：所述待检物为具有免疫活性的蛋白质或与蛋白质偶联产生免疫活性的物质；
    所述待检物特异性结合物为与所述待检物特异性结合的抗原或抗体；
    所述检测指示剂为胶体金属、染料、荧光素和化学发光物质中的至少一种。
  8. 根据权利要求7所述的方法，其特征在于：所述胶体金属为胶体金、胶体硒和胶体金磁微粒中的至少一种；
    所述荧光素为异硫氰酸荧光素、四乙基罗丹明、四甲基异硫氰酸罗丹明、藻红蛋白、多甲藻黄素叶绿素蛋白、碘化丙啶、别藻青蛋白和铕化合物中的至少一种；
    所述化学发光物质为鲁米诺和异鲁米诺及其衍生物类、吖啶酯及吖淀酰胺类、(金钢烷)-1,2-二氧乙烷及其衍生物和三联吡啶钌中的至少一种。
  9. 根据权利要求1所述的方法，其特征在于：所述方法中，所述复合物形成有如下任一种：
    1)所述待检物特异性结合物包含有同时与所述待检物和所述检测相形成特异性结合的一级中间相；
    2)所述待检物特异性结合物包含有同时与所述一级中间相和所述检测相形成特异性结合的二级中间相；
    所述一级中间相和所述二级中间相均为具有特异性结合能力的抗原、抗体、亲和素、生物素及其类似物中的至少一种；
    所述方法中还包括在所述结合反应之后用清洗相清洗所述固相膜的步骤，所述清洗相为含有表面活性剂剂的磷酸盐缓冲液、碳酸盐缓冲液和Tris缓冲液中的至少一种。
  10. 权利要求1所述的方法在免疫产品的含量检测中的应用。
  11. 一种离心分离检测装置，其特征在于：它包括进样部件、固相膜、离心装置和检测器；
    所述离心装置包括由驱动电机驱动的离心转子和支撑底座，所述离心转子以所述支撑底座为支撑；
    所述进样部件设于所述离心转子的中部，并与离心转子相连接；所述进样部件包括液相储存装置和进样管；所述液相储存装置与所述进样管相连通；
    所述固相膜设于所述离心转子上，与所述进样管相连接；
    所述检测器设于所述固相膜的一侧或两侧。
  12. 根据权利要求11所述的离心分离检测装置，其特征在于：所述固相膜置于固相膜放置装置中，且所述固相膜放置装置设于所述离心转子上；
    所述固相膜与所述离心转子为可拆装式结构。
  13. 根据权利要求12所述的离心分离检测装置，其特征在于：所述固相膜放置装置包括旋转移动式固定装置和/或槽形挤压式固定装置。
  14. 根据权利要求11所述的离心分离检测装置，其特征在于：所述固相膜通过固相膜固定器固定，所述固相膜固定器选自固相膜支撑底片样部件、侧向流试纸条扣卡样部件和透明质上下包埋式部件中至少一种。
  15. 根据权利要求14所述的离心分离检测装置，其特征在于：所述透明质上下包埋式部件为所述固相膜的上下两侧由硬质透明材料覆盖，所述固相膜对应侧的所述硬质透明材料的面积大于或等于所述固相膜的面积。
  16. 根据权利要求11所述的离心分离检测装置，其特征在于：所述固相膜在与所述进样管相连的一端还设有与之相连通的液体吸附分散部件，所述固相膜于所述离心转子的远心端设有与之相连通的液体收集部件，所述液体吸附分散部件与所述进样管相连通。
  17. 根据权利要求11所述的离心分离检测装置，其特征在于：所述进样部件包含有进样泵，所述进样泵驱动所述液相储存装置中的液体进入所述进样管。
  18. 根据权利要求17所述的离心分离检测装置，其特征在于：所述液相储存装置包括待检样品储存装置和检测相储存装置；
    所述待检样品储存装置和所述检测相储存装置均与所述进样管相连通，且均由所述进样泵驱动。
  19. 根据权利要求18所述的离心分离检测装置，其特征在于：所述液相储存装置还包括清洗液储存装置，所述清洗液储存装置与所述进样管相连通，且由所述进样泵驱动。
  20. 根据权利要求11所述的离心分离检测装置，其特征在于：所述离心转子采用平面型或由中心向外倾斜型；
    所述离心装置设有一外壳。
  21. 根据权利要求20所述的离心分离检测装置，其特征在于：所述旋转移动式固定装置为在所述离心转子上设置的柱状凸起的连接装置，所述固相膜固定器上设有与所述柱状凸起的连接装置相匹配的孔状部件。
  22. 根据权利要求11所述的离心分离检测装置，其特征在于：所述离 心装置、所述进样部件和所述检测器均设有程序控制装置。
  23. 根据权利要求11所述的离心分离检测装置，其特征在于：所述进样管和所述固相膜采用可拆装式相连接；
    所述液体吸附分散部件与所述进样管采用可拆装式相连通。
  24. 根据权利要求11所述的离心分离检测装置，其特征在于：所述固相膜的材料采用硝酸纤维素膜、聚偏氟乙烯膜、尼龙膜和DEAE纤维素膜中的任一种，且所述固相膜的一面或两面设有背衬；
    所述液体吸附分散部件包括胶体金标记吸附膜、荧光标记抗体吸附膜、化学发光标记吸附膜、多聚酯纤维分散膜和玻璃纤维分散膜中的至少一种；
    所述检测器包括吸光度、荧光、化学发光和图像数字处理的检测器中的任一种。
  25. 根据权利要求11所述的离心分离检测装置，其特征在于：所述离心转子的平面中间段设有孔和/或设有透明部件，且所述孔和/或所述透明部件均使所述固相膜直接暴露于所述检测器。
  26. 权利要求11所述的离心分离检测装置在免疫产品检测中的应用。
  27. 根据权利要求26所述的应用，其特征在于：所述免疫产品包括抗原、抗体、免疫细胞和化学成分中至少一种。
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