WO2018177445A1 - Centrifugation immunochromatography detection method and apparatus - Google Patents

Abstract

Disclosed are a centrifugation immunochromatography detection method and apparatus. The method is based on existing immunochromatographic technologies, utilises a centrifugal apparatus to drive a liquid phase to flow in a solid phase detection film, and may be used for a colloid metal immunochromatography detection method, a fluorescent immunochromatography detection method and a chemiluminescent immunochromatography detection method. The present invention has the features of high sensitivity, short detection time, convenient usage, high stability and convenient storage. Also provided is a lateral flow chromatography detection reaction start control method, wherein a liquid phase loading lateral flow chromatography detection structure is arranged on a centrifugal apparatus, and a liquid phase is driven by centrifugation to enter a solid phase detection film and continue flowing, so as to start a lateral flow chromatography detection reaction. The method may be used for a colloid metal immunochromatography detection method, a fluorescent immunochromatography detection method and a chemiluminescent immunochromatography detection method. The present invention has the features of high accuracy, good reproducibility, convenient usage, high stability and convenient storage.

Description

Centrifugal separation immunochromatographic detection method and device Technical field

The invention relates to a centrifugal separation immunochromatographic detection method and device, and belongs to the technical field of immunodetection.

Background technique

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 antigen or antibody and a luminescent reagent labeling technique for antigen or antibody, but the detection reaction time is long, the detection reagent needs to be refrigerated and transported, and the requirements for the detection equipment are highly affected. Immunofluorescence labeling, flow cytometry, and colloidal gold techniques are also widely used, but they all have their corresponding shortcomings. The long reaction time or lack of sensitivity, accuracy and stability are common deficiencies. High sensitivity, rapidity, convenience, miniaturization, full quantification, and automation are the current development trends of clinical immunoassay technology products, but the existing functions cannot simultaneously achieve the above functions. The automatic control of lateral flow chromatography detection is an important part of the above detection function, and how to artificially control the reaction initiation is the key technology to realize the automation of lateral flow chromatography detection. The prior art of lateral flow detection detects the reaction immediately after loading the liquid, and cannot be started by human control, which severely limits the automation of the batch detection reaction. Therefore, it is important to develop the reaction initiation control technology and method.

Summary of the invention

The object of the present invention is to provide a centrifugal separation immunochromatography detection method and device; the invention has the characteristics of high sensitivity, short detection time, convenient use, high stability and convenient storage.

The invention provides a centrifugal separation detecting method, comprising the steps of: driving the liquid phase in the centrifugal phase detecting membrane by using the centrifugal mechanism in the centrifugal separation detecting device to perform immunochromatography;

The immunochromatography comprises colloidal metal immunochromatography using a colloidal metal as an indicator, fluorescence immunochromatography using fluorescein as an indicator, and chemiluminescent substance and/or chemiluminescent enzyme-mediated luminescence as an indicator of chemiluminescence immunity. Chromatography.

In the above centrifugal separation detecting method, the colloidal metal is at least one of colloidal gold, colloidal selenium, and colloidal gold magnetic particles;

The fluorescein is fluorescein isothiocyanate, tetraethyl rhodamine, rhodamine tetramethyl isothiocyanate, phycoerythrin, polydatin chlorophyll protein, propidium iodide, allophytoin and At least one of the hydrazine compounds;

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 chemiluminescent enzyme is at least one of horseradish peroxidase, alkaline phosphatase, and xanthine oxidase.

In the above centrifugation detection method, the chemiluminescent substrate of the horseradish peroxidase is commonly used for luminol and isoluminol and its derivatives, such as isoluminol, 4-aminohexyl-N-B. Kei Lunuo and AHEI and ABEI, etc., commonly used products are West Pico chemiluminescence detection substrate produced by PIERCE, West Dura chemiluminescence detection substrate, West Femto chemiluminescence detection substrate. Commonly used in the chemiluminescent substrate of alkaline phosphatase are (gold alkane)-1,2-dioxyethane and its derivatives, AMPPD, CDP-STAR, and Lumi-Phos 530. The chemiluminescent substrates of xanthine oxidase are astragalus, myricetin and quercetin.

The above centrifugation detection method further includes a non-enzymatic chemiluminescent substrate, that is, a direct chemiluminescent substance, which is an immunoassay method for directly labeling an antigen or an antibody with a chemiluminescent agent. The commonly used chemiluminescent substance is an acridine ester compound-acridinium ester (AE), which is an effective luminescent label, which emits light by the action of activating luminescent reagent (NaOH, H ₂ O ₂ ), mainly acridinium ester and Amide amides, terpyridines, and the like.

In the above centrifugal separation detecting method, the centrifugal separation detecting method uses microparticles as a carrier carrier of the indicator; the carrier carrier carries the indicator in a manner of using a specific substance to be detected directly labeled by the indicator Directly binding the analyte or the analyte-specific indirect conjugate to label the microparticle or directly labeling the analyte-specific direct conjugate or the analyte-specific indirect conjugate with the microparticle with the indicator;

The microparticles are particles capable of forming non-specific binding to proteins and/or the indicator directly and/or by chemical crosslinking and maintaining stability;

The particle size of the microparticles is 1 nm to 1 um;

The specific conjugate includes an antigen, an antibody, avidin, biotin or a derivative thereof.

In the above centrifugal separation detecting method, the liquid phase includes a liquid phase containing a test substance, a liquid phase of a test substance labeled with the indicator, a liquid phase of a test substance labeled with the fine particles, and a non-marking detection. a liquid phase of the substance, one of the cleaning liquid phases, or a combination thereof;

The test substance is a test substance specific conjugate and a secondary or tertiary specific conjugate thereof;

The specific conjugate includes an antigen, an antibody, avidin, biotin or a derivative thereof.

In the above centrifugal separation detecting method, the centrifugal separation detecting method further includes a step of blocking by using a solid phase detecting membrane blocking liquid;

The blocking solution is a buffer salt solution containing bovine serum albumin and at least one of other soluble protein, skim milk powder, polyethylene glycol, casein, sucrose, surfactant, gelatin, serum, plasma;

The steps of the closing are as follows:

1) coating the solid phase detecting membrane with a solid phase detecting membrane coating liquid, wherein the solid phase detecting membrane coating liquid is a solution of an antigen, an antibody, avidin, biotin or a derivative thereof;

2) washing the solid phase detecting membrane with a washing liquid, which is water, a conventional buffer or a buffer solution containing a surfactant;

3) immersing the solid phase detecting film with the solid phase detecting film blocking liquid;

4) washing the solid phase detecting film again with the cleaning liquid;

5) Dry and spare.

The present invention also provides a lateral flow chromatography detection reaction initiation control method, comprising the steps of: placing a lateral flow chromatography detection mechanism on the centrifugal turntable in the centrifugal separation detecting device, driving by centrifugal driving The liquid phase enters the solid phase detection membrane and maintains the flow, thereby initiating lateral flow chromatography detection analysis;

The lateral flow chromatography detecting mechanism comprises a liquid phase bearing structure and a solid phase detecting film disposed on the supporting film, and the liquid phase bearing structure and the solid phase detecting film are left between the solid phase detecting film and the natural flow of the liquid phase. a gap; the liquid phase bearing structure is located at a proximal end of the solid phase detecting membrane.

In the above lateral flow chromatography detection reaction initiation control method, the colloidal metal is at least one of colloidal gold, colloidal selenium and colloidal gold magnetic particles;

The fluorescein is fluorescein isothiocyanate, tetraethyl rhodamine, rhodamine tetramethyl isothiocyanate, phycoerythrin, polydatin chlorophyll protein, propidium iodide, allophytoin and At least one of the hydrazine compounds;

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 chemiluminescent enzyme is at least one of horseradish peroxidase, alkaline phosphatase, and xanthine oxidase.

In the above lateral flow chromatography detection reaction initiation control method, the centrifugal separation detection method or the lateral flow chromatography detection analysis uses microparticles as a carrier carrier of the indicator; the carrier carrier carries the indicator Means using the analyte-specific direct conjugate or the analyte-specific indirect conjugate directly labeled with the indicator to label the microparticle or directly labeling the microparticle with the indicator directly Sexual direct conjugate or analyte-specific indirect conjugate;

The microparticles are particles capable of forming non-specific binding to proteins and/or the indicator directly and/or by chemical crosslinking and maintaining stability;

The particle size of the microparticles is 1 nm to 1 um;

The specific conjugate includes an antigen, an antibody, avidin, biotin or a derivative thereof.

In the above lateral flow chromatography detection reaction initiation control method, the liquid phase includes a liquid phase containing a test substance, a liquid phase of a test substance labeled with the indicator, and a liquid of a test substance labeled with the fine particles. a liquid phase of a phase, non-labeled test substance, one of a cleaning liquid phase, or a combination thereof;

The test substance is a test substance specific conjugate and a secondary or tertiary specific conjugate thereof;

The specific conjugate includes an antigen, an antibody, avidin, biotin or a derivative thereof.

In the above lateral flow chromatography detection reaction initiation control method, the centrifugal separation detection method further comprises the step of blocking by using a solid phase detection membrane blocking solution;

The blocking solution is a buffer salt solution containing bovine serum albumin and at least one of other soluble protein, skim milk powder, polyethylene glycol, casein, sucrose, surfactant, gelatin, serum, plasma;

The steps of the closing are as follows:

1) coating the solid phase detecting membrane with a solid phase detecting membrane coating liquid, wherein the solid phase detecting membrane coating liquid is a solution of an antigen, an antibody, avidin, biotin or a derivative thereof;

2) washing the solid phase detecting membrane with a washing liquid, which is water, a conventional buffer or a buffer solution containing a surfactant;

3) immersing the solid phase detecting film with the solid phase detecting film blocking liquid;

4) washing the solid phase detecting film again with the cleaning liquid;

5) Dry and spare.

In the above lateral flow chromatography detection reaction initiation control method, the gap is an air or a filter membrane pad;

The gap has a width of 0.5 to 3 mm;

The filter membrane mat has a pore diameter of 0.1 to 5 μm;

The liquid phase bearing structure is a polypolyester fiber membrane, a glass cellulose membrane, a colloidal gold marker-specific sample pad or a fluorescent marker-specific sample pad.

The steps of the above lateral flow chromatography detection analysis and immunochromatography are basically the same.

The invention also provides a centrifugal separation detecting device, comprising a sampling mechanism and a centrifugal mechanism;

The centrifugal mechanism includes a centrifugal rotating disc driven by a driving motor and a supporting base; the centrifugal rotating disc is disposed on the supporting base;

a solid phase detecting film is fixed on the centrifugal rotating disc;

The injection mechanism includes a liquid phase storage device, a sample tube, and a sample pump, the liquid phase storage device is in communication with the sample tube; the sample pump drives liquid in the liquid phase storage device to enter The sample tube;

The sample tube loads a liquid in the liquid phase storage device to a proximal end of the solid phase detection membrane.

In the above centrifugal separation detecting device, the solid phase detecting film is disposed along a radial direction of the centrifugal rotating disk, and is detachably fitted with the centrifugal rotating disk;

The two ends of the solid phase detecting membrane are respectively matched with the liquid adsorption dispersing member and the liquid collecting member;

The liquid adsorption dispersing member is disposed at a proximal end of the centrifugal rotating disc, and the liquid collecting member is disposed at a telecentric end of the centrifugal rotating disc;

The liquid adsorption dispersion component is a liquid phase sample loading site;

The liquid collecting member is for collecting a liquid.

In the centrifugal separation detecting device described above, the material of the solid phase detecting film is any one of a nitrocellulose membrane, a PVDF membrane, a polyvinylidene fluoride membrane, a nylon membrane, and a DEAE cellulose membrane;

The solid phase detecting film is provided with a backing on one or both sides;

The liquid adsorption dispersion member is at least one of a colloidal gold labeled adsorption membrane, a fluorescently labeled antibody adsorption membrane, a chemiluminescent label adsorption membrane, and a dispersion membrane;

The liquid collecting member is a liquid collecting container or made of a water-absorbent material such as absorbent paper and/or water-absorbent gel.

In the above centrifugal separation detecting device, the solid phase detecting film is fixed by a solid phase detecting film fixing device;

The solid phase detecting film fixing device is a snap-like structure, and a sample sampling groove, an observation window and a liquid collecting member outlet are sequentially arranged from one end to the other end;

After the solid phase detecting film is fixed to the solid phase detecting film fixing device, the spotting groove, the observation window, and the liquid collecting member outlet are sequentially connected to the liquid collecting member, the solid phase detecting film, and The liquid collecting member corresponds.

In the above centrifugal separation detecting device, the solid phase detecting film is fixed by a solid phase detecting film fixing device;

The solid phase detecting film fixing device comprises a hard transparent lower cover sheet and a transparent transparent upper cover sheet, and the solid phase detecting film is disposed between the hard transparent lower cover sheet and the transparent transparent upper cover sheet;

One end or both ends of the solid phase detecting film is a hollow interlayer;

During the detection, the liquid phase is centrifuged, passes through the hollow interlayer of the near-heart end, enters the liquid adsorption dispersion member, flows through the solid phase detection membrane, and the liquid phase after the reaction is from the hollow sandwich or the liquid at the telecentric end. The collection parts are discharged.

In the above centrifugal separation detecting device, the centrifugal separation detecting device further includes an ultrasonic breaking device including an ultrasonic generator, a transducer, a horn, and an ultrasonic breaking container which are sequentially connected;

The ultrasonic breaking device is disposed at an upper portion of the solid phase detecting film;

When the ultrasonic disrupting device is used, the horn is lowered, the detection region of the solid phase detecting film is cut, pushed into the ultrasonic breaking container, ultrasonically broken, and then detected. In order to control the controllability and consistency of cutting and crushing, the sonication device is coupled to a component having a programmed speed.

The centrifugation detecting device further includes a detector for detecting a liquid in the solid phase detecting film;

The detector is one or a combination of a colloidal gold quantitative detector, a fluorescence detector, and a chemiluminescence detector;

The detection index of the detector is any one of absorbance, fluorescence value, chemiluminescence value, and image digital signal value or a combination thereof.

In order to control the speed of the centrifugal turntable and the injection speed of the sample introduction mechanism, the centrifugal turntable and the sample introduction mechanism are both connected to a component having a program control speed; the rotation speed of the centrifugal device may be 200~ 10000 r / min, specifically 500 ~ 5000 rev / min, 800 ~ 3000 rev / min or 800 ~ 2000 rev / min.

The centrifugal separation detecting device of the present invention is applied to an immunoassay.

The centrifugal separation detecting device of the present invention can be used in the above-described centrifugal separation detecting method and lateral flow chromatography detecting reaction starting control method.

DRAWINGS

Fig. 1 is a schematic view showing the structure of a centrifugal separation detecting device of the present invention.

2 is a schematic view showing the structure of a solid phase film detecting support film-like member in the centrifugal separation detecting device of the present invention.

Fig. 3 is a schematic view showing the structure of a holder for a solid phase detecting film in the centrifugal separation detecting device of the present invention.

Fig. 4 is a view showing the positional intention of the solid phase detecting film and the injection structure in the centrifugal separation detecting device of the present invention.

Fig. 5 is a structural schematic view showing the solid-phase detecting film holder of the solid-phase detecting film holder in the centrifugal separation detecting device of the present invention.

Fig. 6 is a schematic view showing the position of a solid phase detecting film and an ultrasonic crushing device in the centrifugal separation detecting device of the present invention.

Figure 7 is a schematic illustration of a lateral flow tomography detection structure incorporating a startup control gap structure of the present invention.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Embodiment 1. Horizontal type centrifugal separation detecting device

FIG. 1 is a schematic structural view of a centrifugal separation detecting device according to the present invention, which comprises: a sample introduction mechanism 1, a solid phase detection film 2, a centrifugal device (centrifugal turntable 3, a drive motor 5, a support base 6), and a detector 4; Also included is a sonicator 7 and a housing 8. The sample introduction mechanism 1 corresponds to the proximal end of the solid phase detection film 2, and the ultrasonication device 7 corresponds to the detection portion of the solid phase detection film 2, the centrifugal device includes a centrifugal turntable 3, a drive motor 5 and a support base 6, and the drive motor 5 is located On the support base 6, the centrifugal turntable 3 is driven to rotate by the drive motor 5. The detector 4 is one or a combination of a colloidal gold quantitative detector, a fluorescence detector, and a chemiluminescence detector, and the detection index of the detector 4 is any one of absorbance, fluorescence value, chemiluminescence value, and image digital signal value. Kind or a combination thereof.

As shown in FIG. 1 and FIG. 2, the solid phase detecting film 2 is provided at the proximal end of the centrifugal turntable 3 with a liquid adsorption dispersing member 9 communicating therewith, and the solid phase detecting film 2 is provided at the telecentric end of the centrifugal turntable 3. In connection with the liquid collecting member 10, the solid phase detecting film 2 is fixedly placed on the outer edge of the centrifugal turntable 3 (arranged in the radial direction thereof), and the centrifugal rotating plate 3 is in a detachable structure, and the liquid adsorbing and dispersing member 9 is advanced. The liquid phase sample loading site of the sample mechanism 1. The material of the solid phase detecting film 2 is any one of a nitrocellulose film, a PVDF film, a polyvinylidene fluoride film, a nylon film, and a DEAE cellulose film, and the solid phase detecting film 2 has a back on one or both sides. The liquid absorbing and dispersing member 9 is at least one of a colloidal gold-labeled adsorption film, a fluorescently-labeled antibody adsorption film, a chemiluminescent-labeled adsorption film, and a dispersion film; and the liquid-collecting member 10 is made of a water-absorbent material such as absorbent paper and/or water absorbing material. The gel can also be used as a liquid collection container.

As shown in FIG. 2 and FIG. 3, a solid phase detecting film fixing device for fixing the solid phase detecting film 2, as shown in FIG. 2, supports the backsheet 11, and the supporting film 11 can be selected from a PVC plate, a transparent plastic plate, and an organic glass plate. Wait. The solid phase detecting film fixing device in Fig. 3 is a lateral flow test strip buckle clamping member 12, specifically including a hole member 13, a spotting groove 14, an observation window 15, and a liquid collecting member outlet 16. After the solid phase detecting film structure is placed in the lateral flow test strip buckle-like structure 12, the corresponding portion of the sampling tank 14 is the liquid adsorption and dispersion member 9, and the corresponding portion of the observation window 15 is the solid phase detecting film 2, and the liquid collecting member The corresponding portion of the outlet 16 is the liquid collecting member 10 (which is a water absorbent material or a liquid collecting container).

As shown in FIGS. 1 and 4, the sample introduction mechanism 1 includes a closed phase storage device 17, a liquid phase storage device 18, a sample pump 19, and a sample introduction tube 20. The closed phase storage device 17 and the liquid phase storage device 18 are in communication with the sample introduction tube 20, are disposed above the centrifugal turntable 3, and the liquid phase 21 is driven by the sample pump 19 into the sample introduction tube 20 and then added to the sample sample tank 14 . In order to control the speed of the centrifuge and the injection speed of the sample introduction device, both the centrifuge device and the injection member are connected to a component having a program control speed.

As shown in FIG. 5, another solid phase detecting film fixing device provided for fixing the solid phase detecting film 2 specifically includes a hard transparent lower cover sheet 22, a hard transparent upper cover sheet 24, a front bare empty interlayer 23, and a rear portion. Naked void interlayer or liquid collection component 10. At the time of detection, the liquid phase is centrifuged, passes through the front bare space interlayer 23, enters the liquid adsorption dispersion member 9, flows through the solid phase detection film 2, and the liquid phase after the reaction is discharged from the rear bare space interlayer or the liquid collection member 10.

As shown in FIGS. 1 and 6, the ultrasonic pulverizing device 7 includes an ultrasonic generator 25, a transducer 26, a horn 27, and an ultrasonic breaking container 28, which are located above the solid phase detecting film 2, and when used, the horn 27 When it is lowered, the detection area of the solid phase detecting film 2 is cut, pushed into the ultrasonic breaking container 28, ultrasonically broken, and then detected by a detector. In order to control the controllability and consistency of the cutting and breaking, the centrifugal device, the injection part, the ultrasonic breaking device and the detector are all connected to the part having the program control speed.

Example 2 Comparison of detection correlation between the present invention and current detection methods

First, the experimental materials

Anti-human myoglobin polyclonal antibody (Genagates, USA), anti-human myoglobin monoclonal antibody (Genagates, USA), spectrophotometer (Shanghai Yuhua Technology Instrument Co., Ltd., 752 UV-Vis spectrophotometer), human muscle Red protein (Sigma-Aldrich products), BioFlow printing film instrument (IMAGENE company, USA), Index cutting machine (A-point company, USA), DBF-900 sealing machine (Wenzhou Jiangnan packaging factory), ACBO dehumidifier (Jiangsu Wuxi) Wave dehumidifier company), desktop centrifuge (Eppendoff, USA), bovine serum albumin (abbreviated as BSA, SIGMA product), nitrocellulose membrane (AE 99, supplied by Gengates, USA), polyester cellulose membrane ( Reemay 2033, product of Alstrom, USA), absorbent paper film mat (Grade 470, American S&S company), chloroauric acid (SIGMA product), colloidal gold quantitative chromatography analyzer (Skannex, Norway).

Second, the experimental method

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.

Preparation of colloidal gold-labeled anti-human myoglobin monoclonal antibody: take 10ml of purified water, heat and stir, add 500μl of 10% chloroauric acid solution when the water is boiling, heat and boil for 5 minutes, add 500μl 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. Take a volume of colloidal gold solution 10ml, adjust the pH to 8.3 with 10% potassium carbonate, quickly add 100μg of anti-human myoglobin monoclonal antibody to the final concentration of 10μg/ml, shake the beaker and mix for 30 minutes at room temperature, quickly add 10% 100 ul of bovine serum albumin solution to a final concentration of 1%, while shaking the beaker, standing at room temperature for 30 minutes, centrifuging at 12000 rpm for 20 minutes, carefully aspirating the supernatant; then adding 5 ml of 50 mM phosphate (PBS) buffer, pH 7.4, The pellet was suspended, centrifuged at 12,000 rpm for 20 minutes, and the supernatant was aspirated, and the precipitate was dissolved in 1.0 ml of a phosphate buffer containing 1% of bovine serum albumin and 3% of sucrose, and stored at 4 ° C in the dark.

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 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, and the polyester cellulose membrane was taken. The printing film was set to the conditions of the printing film: the moving speed of the airbrush was 30 mm/sec, and the liquid pushing speed was 3.0 μl/cm. The printed film was placed in a dry box, dried at 37 ° C for 6 hours, and then placed in a desiccant. The sealed bag is stored for use inside.

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 the printing film, set the film conditions: the moving speed of the airbrush is 30mm/sec, and the liquid propelling speed is 0.5μl. /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.

The centrifugal separation detecting device of Example 1 was used for the detection, and the side of the adsorption film labeled with colloidal gold was placed upward, placed in a centrifuge carousel, and a solution of different concentrations of human myoglobin was added to the colloidal gold-labeled adsorption film. After standing for 1 to 15 minutes, 2000 rpm/separation of the heart for 1 minute, and then add 80 ul of 50 mM PBS buffer of pH 7.4 to the colloidal gold-labeled adsorption membrane, and wash it at 2000 rpm for 1 minute, and take out the test strip. The digital image of the polyclonal antibody blotting strip is read on a colloidal gold quantitative chromatography analyzer (ie, a detector), and image processing is performed to obtain a 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 experiment was repeated three times and the results were averaged. Statistically calculate the detection values of different pre-station time test strips.

Third, the experimental results

The measurement result of the detection method using colloidal gold as an indicator shows that the correlation coefficient r2 detected by the technique of the present invention is 0.962, and the correlation coefficient r ² of the prior art detection (not performing centrifugation) is 0.936, P<0.05, remarkable Compared with the detection results of the prior art, the method of the present invention improves the accuracy of the prior art detection. The experimental results are shown in Table 1.

Table 1 Accuracy analysis of detection results using colloidal gold as an indicator (unit: image chromaticity value)

Time (minutes)	technology	3.125	6.25	12.5	25	50	100	r 2
1	Current technology	19.8	19.7	23.0	28.8	31.4	43.7	0.928
	this invention	8.7	7.4	12.3	16.8	24.8	37.9	0.926
2	Current technology	20.3	21.2	23.3	27.6	38.6	44.0	0.932
	this invention	16.5	19.4	26.3	31.8	35.3	39.3	0.965
5	Current technology	34.1	32.1	38.0	42.3	49.1	62.6	0.902
	this invention	23.0	24.3	29.0	38.9	54.8	65.3	0.962
10	Current technology	32.3	31.4	40.1	45.9	61.7	73.8	0.947
	this invention	24.9	29.3	42.3	48.2	61.4	73.1	0.985
15	Current technology	33.2	33.5	43.1	54.3	64.7	76.7	0.972
	this invention	26.1	35.3	45.2	58.1	65.3	74.7	0.971

Example 3, comparison of the minimum detection amount of the present invention and the current detection method

First, the experimental materials

Same as embodiment 2

Second, the experimental method

Same as embodiment 2

Third, the experimental results

In the invention, 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. Results Under the same experimental conditions, the pre-station time was 2-10 minutes, the minimum detection amount in the prior art was 6.25 ng/ml, and the minimum detection amount in the present invention was 3.125 ng/ml, and the detection sensitivity was significantly high. In the prior art, it is explained that the technique of the present invention improves the detection sensitivity of the prior art detection. The experimental results are shown in Table 2.

Table 2 Detection sensitivity analysis of colloidal gold as indicator in the present invention (unit: image chromaticity value)

Example 4, the effect of the invention on detection specificity

First, the experimental materials

Same as embodiment 2

Second, the experimental method

In the same manner as in Example 2, a human myoglobin solution was prepared at 3.125, 6.25, 12.5, 25, 50, 100 ng/ml, and the pre-station time was 5 minutes under the same experimental conditions.

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 taken and the standard curve was detected and drawn using the present invention and the current detection method, respectively.

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. Prepare with sample dilution buffer.

Third, the experimental results

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 value of the repeated detection of the myoglobin sample by the prior art and the present invention is 51 and 49 ng/ml, respectively, and the others do not contain The detection value of the myoglobin sample was below the lower limit of the detection sensitivity of the detection method, and all were negative, and there was no obvious color reaction. It is indicated that the present invention does not affect the detection specificity.

Table 3 Comparison of the results of the present invention and the current technology for detecting myoglobin specificity (unit: ng/ml)

sample	technology	Repeat 1	Repeat 2	Repeat 3	average value
A	Current technology	58	52	43	51

	this invention	55	48	44	49
B	Current technology	<6.25	<6.25	<6.25	<6.25
	this invention	<3.125	<3.125	<3.125	<3.125
C	Current technology	<6.25	<6.25	<6.25	<6.25
	this invention	<3.125	<3.125	<3.125	<3.125
D	Current technology	<6.25	<6.25	<6.25	<6.25
	this invention	<3.125	<3.125	<3.125	<3.125
E	Current technology	<6.25	<6.25	<6.25	<6.25
	this invention	<3.125	<3.125	<3.125	<3.125

Example 5, comparison of detection performance between the present invention and current chemiluminescence detection methods

First, the experimental materials

Anti-human myoglobin polyclonal antibody (Genagates), horseradish peroxidase-labeled anti-human myoglobin monoclonal antibody (Genagates, USA), magnetic particles (MP-COOH-20020, Zhengzhou Yingnuo Biotechnology Co., Ltd. ), pico luminescent reagent (Thermo scientific), chemiluminescence detector (Promega, Glomax Multi JR Detection System), human myoglobin (Sigma-Aldrich products), BioFlow printing film instrument (IMAGENE, USA), Index cutting machine ( American A-point company), DBF-900 sealing machine (Wenzhou Jiangnan Packing Factory), ACBO dehumidifier (Jiangsu Wuxi Aobo Dehumidifier Company), desktop centrifuge (Eppendoff Company, USA), bovine serum albumin (SIGMA product), Nitrocellulose membrane (AE 99, supplied by Genagates, USA), polyester cellulose membrane (Reemay 2033, product of Alstrom, USA), absorbent paper membrane mat (Grade 470, American S&S company product).

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 2.

1. Current chemiluminescence detection method group

For magnetic particle labeling, 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 μl of magnetic particles labeled with anti-human myoglobin polyclonal antibody were added to each tube, and 100 μl of each concentration of human myoglobin solution was added to each concentration, and the binding reaction was shaken at 37 ° C. After incubating for 60 minutes, the magnetic particles were separated by magnetic separation, the supernatant was discarded, washed twice with 200 μl of PBS, the magnetic particles were separated by magnetic separation, the supernatant was discarded, and horseradish peroxidase-labeled anti-human myoglobin was added. 200 μl 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 μl 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 μl 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.

2. The invention group

The polyester polyester film was pretreated as in Example 2, dried at 37 ° C, and sealed for use.

The unsealed group polyclonal antibody printing film of the present invention is stored in the same manner as in Example 2 and placed in a desiccant-containing dry container.

The closed group polyclonal antibody printing film of the present invention was subjected to the same treatment as in Example 2, and the printed film was placed in a 37 ° C dry box and dried for 1 hour. Prepare a blocking solution (5% skim milk powder, 3% BSA, 0.05% tween20, 0.8% NaCl, 0.02% KCL, pH 7.4), place the treated polyclonal antibody print in a blocking solution, and let stand at room temperature for 30 minutes. It was taken out and placed in a 37 ° C dry box and dried for 6 hours.

Preparation of Chemiluminescent Labeled Adsorption Membrane: Pretreated Polyester Cellulose Membrane, Dilute Horseradish Peroxidase Labeled Anti-Human Myoglobin Monoclonal Antibody 0.15mg/ml with 50mM PBS Buffer (pH 7.4), Start The film printer, loading the antibody, taking the polyester cellulose film, starting the printing film, setting the film conditions: the moving speed of the airbrush is 30 mm/sec, the liquid pushing speed is 3.0 μl/cm, and the printed film is freeze-dried. Store at 4 ° C for storage.

Semi-finished product assembly method: start the dehumidifier to reduce the humidity in the operating room to less than 25%, and paste the absorbent paper film pad and the chemiluminescent label 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.

Using the centrifugal separation detecting device of the first embodiment of the present invention, the test strip prepared above is taken, and the side of the chemiluminescence-labeled adsorption film is placed upward, placed in the centrifuge carousel, and the composition is added dropwise to the chemiluminescent label adsorption film. The concentration of human myoglobin solution was 80 ul, allowed to stand for 3 minutes, 2000 rpm/separation of the heart for 1 minute, and then add 7.4 buffer of PBS buffer containing 1% Tween 20 at pH 7.4 to the chemiluminescent label adsorption membrane, 2000 rpm. / Separation of the heart for 1 minute cleaning, repeated washing 2 times, remove the test strip, cut the polyclonal antibody film detection line, placed in a transparent test tube, add 200 ul of PBS buffer, ultrasonication for 3 seconds. Take 10ul, add Pico luminescent reagent, put it on the chemiluminescence detector, record the luminescence amount when the reaction is carried out for 2 minutes, the luminescence integration time is 6 seconds, the experiment is repeated three times, the result is averaged, then the concentration-luminescence curve is drawn, and Calculate the correlation coefficient.

Third, the experimental results

The human myoglobin solution is detected by the chemiluminescence detection method and the current chemiluminescence detection method, 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 r2 value The correlation coefficient r2 was 0.95 and 0.901, respectively, but the unclosed group had a correlation coefficient r2 of 0.75. It is indicated that the closed group of the present invention has a detection effect similar to the current chemiluminescence technology, but the detection time is significantly shortened, and the background signal of the unclosed group is high, which affects the detection.

Table 4 Comparison of the detection performance of the present invention and the current chemiluminescence detection method (unit: luminescence value)

Concentration (ng/ml)	The invention is closed	current technology	The invention is not closed
3.125	95380	661292	1161292
6.25	112336	783025	1283025
12.5	329378	821436	1121436
25	956238	1123185	1323185
50	1608326	2234590	2420395
100	3082607	3411312	3518320
r 2 value	0.974	0.901	0.750

Example 6, the microsphere-mediated chemiluminescence detection experiment of the present invention

Fluorescent microspheres are used as detection reaction carriers.

First, the experimental materials

Fluorescent microspheres (Shanghai Jieyi Bio), Trehalose (SIGMA), nitrocellulose membrane (Millipore), EDC (PIERCE), NHS (PIERCE), horseradish peroxidase-labeled anti-human myoglobin Cloned antibody (Genagates, USA), anti-human myoglobin polyclonal antibody (Genagates, USA), spectrophotometer (Shanghai Hanhua Scientific Instrument Co., Ltd., 752 UV-Vis spectrophotometer), human myoglobin (Sigma-Aldrich) Products), BioFlow printing film instrument (IMAGENE company, USA), Index cutting machine (A-point company, USA), DBF-900 sealing machine (Wenzhou Jiangnan packaging factory), ACBO dehumidifier (Jiangsu Wuxi Aobo dehumidifier company), Benchtop centrifuge (Eppendoff, USA), bovine serum albumin (abbreviated as BSA, SIGMA), nitrocellulose membrane (AE 99, supplied by Gengates, USA), polyester cellulose membrane (Reemay 2033, Alstrom, USA) Product), absorbent paper film mat (Grade 470, US S&S company product), chemiluminescence detector (Promega, Glomax Multi JR Detection System), West Pico luminescent reagent (Thermo scientific).

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 2.

Fluorescent microsphere labeling: Take 0.5ml microspheres, centrifuge 4 times with 0.1M phosphate buffer pH6.2, centrifuge at 13000rpm, reconstitute to 1ml with 0.1M phosphate buffer pH6.2, add 2mg horseradish peroxidation. Enzyme-labeled anti-human myoglobin monoclonal antibody, mix, add 250ul of 40mg/ml EDC solution, add 250ul 40mg/ml NHS solution, mix, react at room temperature for 60 minutes, add 20mg of bovine serum albumin, Mix and react at room temperature for 60 minutes. The supernatant was centrifuged, washed 4 times with 0.05 M Tris pH 7.6, reconstituted to 10 ml with 0.5% trehalose, 1% BSA, 0.05 M Tris pH 7.6, and stored at 4 ° C in the dark.

Preparation of fluorescent microsphere-labeled adsorption membrane: The fluorescent microsphere-labeled antibody solution was diluted 3 times with the complex solution, and the other printing method in the preparation of the colloidal gold-labeled adsorption membrane of Example 2 was used.

Polyclonal antibody print preparation: same as in Example 2.

Semi-finished product assembly method: same as in the second embodiment.

Experimental group: Take the test strip prepared above, and mark the side of the adsorption membrane with the fluorescent microspheres upwards, place it in the centrifuge turntable, and add different concentrations of human myoglobin to the fluorescent microsphere labeling adsorption membrane. 80 ul of solution, let stand for 3 minutes, 1000 rpm / separation of the heart for 1 minute, and then add 0.05 mM Tween-20, 50 mM PBS buffer, 80 ul, 2000 rpm to the fluorescent microsphere labeled adsorption membrane. After washing for 30 seconds, wash twice, take out the test strip, cut out the polyclonal antibody test line, place it in a transparent test tube, add 200 ul of PBS buffer, and sonicate for 3 seconds. Take 10ul, add Pico luminescent reagent, put it on the chemiluminescence detector, record the luminescence amount when the reaction is carried out for 2 minutes, the luminescence integration time is 6 seconds, the experiment is repeated three times, the result is averaged, then the concentration-luminescence curve is drawn, and Calculate the correlation coefficient.

Control group: Take the test strip prepared above, place it on the table top, add 80ul of different concentrations of human myoglobin solution to the fluorescent microsphere labeling adsorption membrane, and let it stand. The fluorescent microspheres mark the liquid on the adsorption membrane. All the chromatograms were flowed into the nitrocellulose membrane, and then 0.05 μm of Tween-20 and 50 mM PBS buffer solution of pH 7.4 were added to the fluorescent microsphere-labeled adsorption membrane, and the mixture was allowed to stand. The liquid was all flowed into the nitrocellulose membrane, and the two were washed. Next, remove the test strip, cut the polyclonal antibody print test line, place it in a transparent test tube, add 200 ul of PBS buffer, and sonicate for 3 seconds. Take 10ul, add Pico luminescent reagent, put it on the chemiluminescence detector, record the luminescence amount when the reaction is carried out for 2 minutes, the luminescence integration time is 6 seconds, the experiment is repeated three times, the result is averaged, then the concentration-luminescence curve is drawn, and Calculate the correlation coefficient.

Third, the experimental results

In this experiment, the fluorescent microspheres were used as the liquid phase reaction carrier for detection. As a result, the average detection time of the test strips of the experimental group of the present invention was 4.6 minutes, and the test strips of the control group (without centrifugation) were single-detected. The average time is 49 minutes; the experimental data of the experimental group of the present invention has a good correlation of the concentration-luminescence value, the correlation coefficient r ² is 0.987, and the control data of the control group is below 50 ng/ml, and there is substantially no correlation of the concentration-luminescence value. Mainly because the non-specific binding of the chemiluminescent substance on the solid phase membrane can not be effectively cleaned, which leads to the background being too high, the correlation coefficient r ² is 0.711, P<0.05, and the result of the invention is significantly better than the detection without centrifugation. As a result, it is explained that the technique of the present invention improves the linearity and accuracy of prior art detection. The experimental results are shown in Table 5.

Table 5 Experimental results of immunochromatography chemiluminescence detection of the present invention (unit: luminescence value)

technology	3.125	6.25	12.5	25	50	100	r 2
test group	80235	182278	387126	1232961	1782785	3672463	0.987
Control group	1261453	1288604	1322169	1297865	2321226	4120437	0.711

Example 7 Comparison with the test results of current chemiluminescence detection methods

First, the experimental materials

Same as Embodiment 6.

Second, the experimental method

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 taken, and a standard curve was detected and plotted by the chemiluminescence detection method of the present invention. A known concentration of human myoglobin 30 ng/ml was used as a sample to be tested. The other methods were the same as those in the experimental group of Example 6.

Third, the experimental results

The specific results of the three repeated experiments are shown in Table 6. The measurement result of the centrifugation technique of the invention shows that the content of the human myoglobin of the sample to be tested is 30.95 ng/ml, which has good repeatability and accuracy.

Table 6 Test performance of chemiluminescence detection method of the present invention (unit: ng/ml)

	Repeat 1	Repeat 2	Repeat 3	average value
Centrifugation	28.76	29.55	34.55	30.95

Example 8, comparison of detection performance between the present invention and current fluorescent immunoassay methods

First, the experimental materials

Anti-human myoglobin polyclonal antibody (Genagates), anti-human myoglobin monoclonal antibody (Genagates, USA), fluorescent microspheres (fluorescein used as bismuth compound, Shanghai Jieyi Biotech Co., Ltd.), EDC (Pierce product) , NHS (Pierce products), human myoglobin (Sigma-Aldrich products), fluorescence quantitative analyzer (Shanghai tissue biotechnology company, HG-98), BioFlow printing film instrument (US IMAGENE company), Index cutting machine (USA A -point company), DBF-900 sealing machine (Wenzhou Jiangnan Packing Factory), ACBO dehumidifier (Jiangsu Wuxi Aobo Dehumidifier Company), desktop centrifuge (Eppendoff Company, USA), bovine serum albumin (SIGMA product), nitrocellulose Membrane (AE 99, supplied by Genagates, USA), polyester cellulose film (Reemay 2033, product of Alstrom, USA), absorbent paper film mat (Grade 470, American S&S company).

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 2.

Fluorescent microsphere labeling: Take 0.5ml fluorescent microspheres, centrifuge 4 times with 0.1M PB of PH6.2, centrifuge at 13000 rpm, reconstitute to 1ml with 0.1M PB pH6.2, add 150ug anti-human myoglobin monoclonal The antibody was mixed, 0.1 M PB of pH 6.2 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.

Preparation of fluorescently labeled antibody adsorption membrane: preparing a polyester cellulose membrane pretreatment liquid containing 0.5% PVA, 50 mM PBS solution, 0.5% BSA, 0.88% NaCl, pH 7.4, and treating the polypolyester cellulose film to be treated The pretreatment liquid was immersed for 1 hour at room temperature, the film was taken out, dried at 37 ° C, and sealed for use. 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 polyester cellulose membrane was taken, the film was printed, and the film conditions were set. The spray gun has a moving speed of 30 mm/sec and a liquid advancing speed of 5.0 μl/cm. The printed film is placed in a dry box, dried at 37 ° C for 6 hours, and then stored in a sealed bag containing a desiccant.

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, start printing the film, set the film conditions: the moving speed of the airbrush is 30mm/sec, the liquid propelling speed is 0.5μl/cm, and it will be printed. The 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 chamber to less than 25%, paste the absorbent paper film pad and the fluorescent-labeled antibody 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.

Taking the test strip prepared above, the side of the fluorescent-labeled antibody adsorption membrane is placed upward, placed in a centrifuge carousel, and 80 ul of different concentrations of human myoglobin solution are added to the fluorescent-labeled antibody adsorption membrane, and allowed to stand 3 Minutes, 2000 rpm / separation of the heart for 1 minute, and then add 80 ul of pH 7.4 PBS buffer to the fluorescent labeling antibody adsorption membrane, 2000 rpm / separation of the heart for 1 minute to wash, remove the test strip, read on the fluorescence quantitative analyzer The fluorescence value of the polyclonal antibody blot was taken.

The current technical control test strips were not centrifuged, and after standing for a set period of 3 minutes, they were allowed to stand for another 2.5 minutes, and then the fluorescence value was read.

The experiment was repeated three times and the results were averaged.

Third, the experimental results

The specific results are shown in Table 7. The technique of the present invention was as described above, and the linear reaction was good, and the correlation coefficient r ^{2 was} 0.994. According to the prior art measurement, after standing for 3 minutes, and then standing for 2.5 minutes, 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 ² is 0.900.

Table 7 Comparison of detection performance between the present invention and current immunofluorescence detection methods (unit: luminescence value)

Concentration (ng/ml)	this invention	current technology
3.125	18820	84125
6.25	45323	87873
12.5	97832	92451
25	152371	186782
50	324569	317653
100	632872	587749
r 2	0.994	0.900

Example 9, comparison between the present invention and current immunofluorescence detection methods

First, the experimental materials

Sheep anti-mouse IgG polyclonal antibody (provided by Genagagates, USA), otherwise as in Example 8.

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 2. A 20 ng/ml known concentration of human myoglobin test sample was prepared.

A 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 taken and the standard curve was detected and plotted using the present invention and the prior art, respectively.

Fluorescent microsphere labeling: same as in Example 8.

Printing of fluorescent microsphere membrane: same as in Example 8.

Preparation of fluorescently labeled antibody adsorption membrane: same as in Example 8.

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). A goat anti-mouse IgG polyclonal antibody solution was taken and diluted 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, 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 μl/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 assembly method: same as in Example 8.

Taking the test strip prepared above, the side of the fluorescent labeling antibody adsorption membrane is turned up, placed in a centrifuge carousel, and the prepared human myoglobin solution and the sample to be tested are respectively added to the fluorescent labeled antibody adsorption film. 80 ul, let stand for 3 minutes, 2000 rpm / separate the heart for 1 minute, then add 80 ul of pH 7.4 PBS buffer to the fluorescent labeling antibody adsorption membrane, 2000 rpm / separate the heart for 1 minute to wash, remove the test strip, in the fluorescence The fluorescence value of the detection line T and the quality control line C on the polyclonal antibody printed film was read on a quantitative analyzer, and the T/C ratio was calculated, a standard curve was drawn, and the myoglobin concentration of the sample to be tested was calculated.

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 experiment was repeated three times and the results were averaged.

Third, the experimental results

The technique of the present invention is operated as above, the standard curve is linear, and the correlation coefficient r2 is 0.995, and then the sample is measured, and the average of three experiments is 19.12 ng/ml, which meets the requirements. It was measured by the prior art, and after standing for 3 minutes, it was allowed to stand for 2.5 minutes, and the linearity of the detection standard curve was not good, and the correlation coefficient r ^{2 was} 0.937. Subsequently, the total spotting time was extended to 15 minutes of the current product test. The standard curve was linear and the correlation coefficient r ^{2 was} 0.961. Then the sample was measured according to the conditions of 15 minutes. The three average values were 20.84 ng/ Ml, meets the requirements. The specific results of three replicate experiments are shown in Table 8. Compared with the prior art, the present invention significantly shortens the detection time.

Table 8 Analysis of the detection results of the present invention by immunofluorescence detection method (unit: ng/ml)

	Repeat 1	Repeat 2	Repeat 3	average value
this invention	16.98	21.22	19.17	19.12
current technology	22.35	21.21	18.97	20.84

Example 10 Comparison of the detection results of the fluorescence detection method and the current enzyme-linked immunosorbent assay method of the present invention

First, the experimental materials

Enzyme-linked immunosorbent assay (Bio-Rad, Model 550), healthy human serum (healthy volunteer donation), and the same as in Example 8.

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 2.

A known concentration of 16.1 ng/ml human myoglobin healthy human serum was used as a sample to be tested.

Making a standard curve: taking a known concentration of human myoglobin solution 3.125, 6.25, 12.5, 25, 50, 100 ng / ml human myoglobin solution, using the fluorescence detection of the present invention and the current enzyme-linked immunosorbent assay to detect human muscle red The protein solution is drawn and a standard curve is drawn, and then the sample to be tested is taken, and the concentration of myoglobin in the sample to be tested is calculated using a standard curve. Make 3 parallel tubes for each sample.

1. Current enzyme-linked immunosorbent assay method group

Using 96-well enzyme-linked plates, 100 μl of anti-human myoglobin polyclonal antibody was added to each tube, coated at 4 ° C overnight, washed three times, and then added with human myoglobin solution or 100 μl of the sample to be tested, and the binding reaction was incubated at 37 ° C. After 120 minutes, wash three times, add 100 μl of anti-human myoglobin monoclonal antibody, incubated for 60 minutes at 37 ° C, wash three times, discard the supernatant, add horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody 100 μl The binding reaction was incubated at 37 ° C for 60 minutes, washed three times, the supernatant was discarded, and 100 μl of the color developing solution was added (formulation: 0.1 M citric acid 2.43 ml, 0.2 M disodium hydrogen phosphate 2.57 ml, o-phenylenediamine 5 mg, peroxidation) 5 μl of hydrogen, protected from light for 5 minutes, and quenched by the addition of 2M sulfuric acid. The absorbance of OD490 was read on an enzyme-linked immunosorbent assay, and a standard curve was drawn, r2=0.991, and the content of human myoglobin in the sample to be tested was calculated.

2. The invention group

Fluorescent microsphere labeling: same as in Example 8.

Printing of fluorescent microsphere membrane: same as in Example 8.

Preparation of fluorescently labeled antibody adsorption membrane: same as in Example 8.

Polyclonal antibody print preparation: same as in Example 9.

Semi-finished assembly method: same as in Example 8.

Taking the test strip prepared above, the side of the fluorescent labeling antibody adsorption membrane is turned up, placed in a centrifuge carousel, and the prepared human myoglobin solution and the sample to be tested are respectively added to the fluorescent labeled antibody adsorption film. 80 ul, let stand for 3 minutes, 2000 rpm / separate the heart for 1 minute, then add 80 ul of pH 7.4 PBS buffer to the fluorescent labeling antibody adsorption membrane, 2000 rpm / separate the heart for 1 minute to wash, remove the test strip, in the fluorescence The fluorescence value of the detection line T and the quality control line C on the polyclonal antibody printing film was read on a quantitative analyzer, and the T/C ratio was calculated, and a standard curve was drawn, r ² =0.987, and the myoglobin concentration of the sample to be tested was calculated. .

Third, the experimental results

The specific results are shown in Table 9. The results of the current enzyme-linked immunosorbent assay showed that the content of human myoglobin in the sample to be tested was 16.54 ng/ml. The results of the present invention showed that the content of human myoglobin in the sample to be tested was 16.91 ng/ml, and the results obtained by the two experimental methods were obtained. 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.

Table 9 Comparison of the detection results of the fluorescence detection of the present invention and the current enzyme-linked immunosorbent assay (unit: ng/ml)

	Repeat 1	Repeat 2	Repeat 3	average value
Current technology	18.72	17.28	13.63	16.54
this invention	15.44	18.91	16.37	16.91

Example 11 Effect of Centrifugal Velocity of the Invention on Test Results

First, the experimental materials

Same as Embodiment 2.

Second, the experimental method

In the same manner as in Example 2, it was pre-positioned for 3 minutes.

Third, the experimental results

In the present invention, 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 10. It can be seen from Table 10 that the detection accuracy is related to the centrifugal speed, and the centrifugal speeds of 500, 1000, 2000, 3000, 4000 rpm are obtained to meet the required test results, and the correlation coefficient r ² values are all greater than 0.98. 4000, 5000 rpm The detection results obtained by the centrifugal speed have a correlation coefficient r ^{2 of} less than 0.98, which does not meet the relevant detection requirements. It is indicated that the optimal centrifugation speed for detecting myoglobin of the present invention should be below 3000 rpm.

Table 10 Effect of centrifugal speed on test results

Centrifugal speed	3.125	6.25	12.5	25	50	100	r 2
500r/min	30.2	34.7	40.7	49.2	63.8	78.5	0.988
1000r/min	29.7	32.0	38.9	50.6	61.8	79.8	0.980
2000r/min	28.8	33.5	38.0	46.8	56.4	82.4	0.962
3000r/min	28.1	33.2	37.7	49.2	56.4	74.3	0.987
4000r/min	19.2	24.8	26.6	28.4	34.7	66.9	0.835
5000r/min	19.8	21.5	26.4	28.1	34.2	62.0	0.864

Example 12, the effect of the particle size of the present invention on the detection result

The carrier is reacted with a polystyrene microsphere as a liquid phase reaction.

First, the experimental materials

Polystyrene microspheres (particle size 35, 130, 376, 600, 1000 nm, carboxylated, Shanghai Taoyu International), otherwise the same as in Example 2.

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 2.

Anti-human myoglobin monoclonal antibody FITC labeling: using the Marsshall method, take 3mg/ml anti-human myoglobin monoclonal antibody solution, add 1/10 volume of 0.5M (pH 9.0) carbonate buffer, electromagnetic stirring 5 minutes. A 1 mg/ml FITC solution was prepared in 50 mM phosphate (PBS), pH 8.0 buffer, slowly added to the antibody solution with stirring at 30 ul/ml, and stirred at 4 ° C for 12 hours in the dark. The labeled antibody solution was centrifuged (2500 r/min) for 20 minutes, the precipitate was removed, placed in a dialysis bag, and dialyzed overnight in 50 mM PBS buffer at 4 °C. The dialyzed overnight marker was passed through a Sephadex G-25 column, free FITC was isolated, and the labeled fluorescent antibody, FITC-labeled anti-human myoglobin monoclonal antibody, was collected and stored at -20 ° C in the dark.

Polystyrene microsphere labeling: Take 2ml 0.1M MES, pH5.0 solution, FITC labeled anti-human myoglobin monoclonal antibody 2mg, 0.2ml 5% w/v microspheres and 20mg EDC, mix, add 10mg NHS Place on a rotary mixer shaker for 2 hours at room temperature, centrifuge at 12000 rpm for 15 minutes, discard the supernatant, add 2 ml of 20 mM PBS, 1% BSA in blocking buffer for 1 hour at room temperature, add 4 ml of 50 mM PBS, pH 7.4 buffer. Suspension, repeated washing once, adding 2 ml of 50 mM PBS buffer, and storing at 4 ° C for use.

Preparation of polystyrene microsphere-labeled adsorption membrane: Take polystyrene microsphere-labeled antibody solution, dilute to 1% w/v microspheres with 50 mM PBS, pH 7.4 buffer, start the membrane tester, load antibody, and pressurize Nitrogen, take multi-polyester cellulose film, start printing film, set the film conditions: spray pen movement speed 30mm / sec, liquid propulsion speed 5.0μl / cm, put the printed film into the dry box, 37 Dry at °C for 6 hours, then store in a sealed bag containing desiccant.

Polyclonal antibody print preparation: same as in Example 2.

Semi-finished product assembly method: same as in the second embodiment.

Experimental group: Take the test strip prepared above, and mark the side of the adsorption film with the polystyrene microspheres upwards, place it in the centrifuge turntable, and add the different concentrations of the people to the polystyrene microsphere label adsorption film. 80 μl of myoglobin solution, allowed to stand for 3 minutes, centrifuged at 1000 rpm for 1 minute, and then added 0.05 mM Tween 20, 50 mM PBS buffer 80 ul, 2000 to the polystyrene microsphere labeled adsorption membrane. The core was rotated/separated for 30 seconds, the test strip was taken out, and the fluorescence value was read on a fluorescence detector. The experiment was repeated three times, and the results were averaged, then the concentration-luminescence curve was plotted, and the correlation coefficient was calculated.

Third, the experimental results

The present invention uses polystyrene microspheres as a liquid phase reaction carrier to observe the effect of different particle sizes on the experimental results. As a result, the detection data of the present invention having a particle diameter of 35, 130, 376, 600, and 1000 nm showed a good correlation of the concentration-luminescence value, and the correlation coefficient r ² was greater than 0.98, indicating that particles having different particle diameters were suitable for the technique of the present invention. The experimental results are shown in Table 11.

Table 11 Effect of Particle Size of the Invention on Detection Results (Unit: Luminescence Value)

Particle size

3.125

6.25

12.5

25

50

100

r 2

(nm)
35	83160	113520	225362	285321	391786	450012	0.955
130	95461	168213	213382	242914	322769	394864	0.950
376	109234	156378	223451	267869	297712	403524	0.967
600	94532	134568	212219	278763	318826	398772	0.964
1000	95674	112367	198756	287964	312278	412344	0.956

Example 13, the influence of the cleaning step of the present invention on the detection result

First, the experimental materials

Same as Embodiment 2.

Second, the experimental method

The experimental washing step was allowed to stand without washing. Others are the same as in the second embodiment.

Third, the experimental results

The experimental results are shown in Table 12. Comparing the results of the two experimental groups, the cleaning results were better than the non-cleaning, and the correlation coefficient r ² was 0.968 with 0.943. It is necessary to indicate that the step of washing after the experimental injection is necessary.

Table 12 Effect of the cleaning step of the present invention on the detection result (unit: image chromaticity value)

Concentration (ng/ml)	Cleaning	Non-cleaning
3.125	20.3	29.6
6.25	22.2	28.7
12.5	24.3	33.5
25	31.1	37.1
50	38.6	41.4
100	47.7	48.6
r 2	0.968	0.943

Example 14, Effect of avidin biotin amplification system on the detection performance of the present invention

The colloidal gold particles are used as a liquid phase reaction carrier.

First, the experimental materials

NHS activated biotin (SIGMA), avidin (SIGMA), and the same as in Example 2.

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 2, the concentrations were 0.1, 1, 3.13, 6.25, 12.5, 25, 50, 100 ng/ml.

Anti-human myoglobin polyclonal antibody biotin labeling: Anti-human myoglobin polyclonal antibody was dialyzed against 0.1 M pH 9.5 sodium carbonate buffer overnight to a final concentration of 2 mg/ml. 20 mg of NHS-activated biotin was dissolved in 1 ml of dimethylformamide, and 50 ul was added thereto, and the solution was added to the above solution, and reacted at room temperature for 4 hours. The reaction solution was dialyzed against PBS buffer overnight and stored at -20 °C.

Colloidal gold particle labeling: same as in Example 2.

Preparation of colloidal gold particle-labeled adsorption membrane: Take colloidal gold particle-labeled antibody solution, dilute to OD530 with colloidal buffer (1% BSA, 3% sucrose, 50 mM PBS, pH 7.4), add biotin-labeled anti-human muscle red The protein polyclonal antibody was 10 μg/ml and mixed. Others are the same as in the second embodiment.

Affinity imprinting membrane preparation: Avidin solution was taken and diluted to a concentration of 1 mg/ml with 50 mM phosphate buffer (pH 7.4). The film press was started and the avidin solution was loaded, otherwise the same as in Example 2.

Semi-finished product assembly method: start the dehumidifier to reduce the humidity in the operation chamber to 25% or less, and paste the absorbent paper film pad and the colloidal gold particle-labeled adsorption film on both ends of the avidin printing film, and the same as in the second embodiment.

Experimental group: Take the test strip prepared above, and mark the side of the adsorption film with the colloidal gold particles facing up, place it in the centrifuge turntable, and add the different concentrations of human myoglobin solution to the colloidal gold particle-labeled adsorption film. 80 ul, let stand for 3 minutes, 1000 rpm / separation of the heart for 1 minute, and then add 0.05 mM Tween-20, 50 mM PBS buffer, 80 ul of pH 7.4 to the colloidal gold particle-labeled adsorption membrane, 2000 rpm/separation of heart 30 After cleaning in seconds, the test strip is taken out and placed on a colloidal gold quantitative chromatograph to read the chromaticity value. The experiment is repeated three times, and the result is averaged, then the concentration-chroma value curve is drawn, and the correlation coefficient is calculated.

Control group: Take the test strip prepared above, place it on the table top, add 80ul of different concentrations of human myoglobin solution to the colloidal gold particle-labeled adsorption film, and let it stand, until the red color on the colloidal gold particle-labeled adsorption film The colloidal gold particle label was completely chromatographed into the nitrocellulose membrane, and then 0.05 mM Tween-20 and 50 mM PBS buffer solution of pH 7.4 were added to the colloidal gold particle-labeled adsorption membrane, and allowed to stand, and the liquid was all flowed into the nitrocellulose. The film was removed, and the test strip was taken out and placed on a colloidal gold quantitative chromatography analyzer to read the chromaticity value. The experiment was repeated three times, and the results were averaged, then the concentration-chroma value curve was drawn, and the correlation coefficient was calculated.

Third, the experimental results

In the invention, the colloidal gold particles are used as a liquid phase reaction carrier. As a result, the average detection time of the test strip of the experimental group of the invention is 3.7 minutes, and the average time of the single test of the test strips (without centrifugation) is averaged. It is 38 minutes; the experimental data of the experimental group of the present invention has a good correlation of the concentration-chroma value, the correlation coefficient r ² is 0.973, the linear detection range is 0.1-100 ng/ml, and the correlation detection r ² of the control group is 0.902. The detection requirement that r is greater than 0.98 is not reached, but when the linear detection range is adjusted to 3.125 to 100 ng/ml, the correlation coefficient r ² is 0.973, and the detection requirement of r greater than 0.98 is achieved. The experimental results show that the technique of the present invention not only shortens the detection time, but also improves the sensitivity and accuracy of the prior art detection. The experimental results are shown in Table 13.

Table 13 Effect of avidin biotin amplification system on the detection performance of the present invention (unit: chroma value)

Concentration (ng/ml)	amplification	Not magnified
0.1	10.2	12.6

1	16.4	13.8
3.125	18.6	16.8
6.25	22.8	24.5
12.5	24.5	35.0
25	29.7	38.6
50	40.2	48.2
100	46.8	56.4
r 2	0.973	0.902

Example 15, Separation and Detection Structure for Controlling Liquid Phase Flow by Centrifugal Drive

As shown in FIG. 7, the present invention includes a lateral flow chromatography detection structure for initiating a control gap structure, including a liquid phase bearing structure 29, a gap structure 30, a solid phase detecting film 2, a liquid collecting member 9, and a supporting backsheet 11. When used, the lateral flow chromatography detection structure is placed on the centrifugal turntable 3 of the centrifugal separation detecting device shown in FIG. 1, and the orientation is sequentially the liquid phase bearing structure 29, the gap structure 30, the solid phase detecting film 2, and the liquid collecting member. 9 and supported by the support film 11, the liquid phase bearing structure 29 is located at the proximal end of the centrifugal turntable 3.

In actual operation, the liquid phase is loaded onto the liquid phase carrying structure 29 by the injection mechanism 1, and when it is at rest, due to the gap between the liquid phase bearing structure 29 and the solid phase detecting film 3 which hinders the natural flow of the liquid phase The structure 30 hinders the natural flow of the liquid phase onto the solid phase detecting film 3, and the liquid phase in the liquid phase bearing structure 29 cannot flow into the solid phase detecting film 3, and the detection reaction is in a suspended state. However, when the centrifugation is initiated, the centrifugal force drives the liquid phase to flow through the gap structure 30 into the solid phase detecting membrane 3 and flow forward in the membrane, and initiates lateral flow chromatography to detect the reaction.

Example 16. Comparative test experiment using colloidal gold as an indicator in the present invention

First, the experimental materials

Anti-human myoglobin polyclonal antibody (Genagates, USA), anti-human myoglobin monoclonal antibody (Genagates, USA), spectrophotometer (Shanghai Yuhua Technology Instrument Co., Ltd., 752 UV-Vis spectrophotometer), human muscle Red protein (Sigma-Aldrich products), BioFlow printing film instrument (IMAGENE company, USA), Index cutting machine (A-point company, USA), DBF-900 sealing machine (Wenzhou Jiangnan packaging factory), ACBO dehumidifier (Jiangsu Wuxi) Wave Dehumidifier Company), Benchtop Centrifuge (Eppendoff, USA), Bovine Serum Albumin (BSA, SIGMA), Nitrocellulose Membrane (AE99, supplied by Gengates, USA), Polyester Cellulose Membrane (Reemay) 2033, Alstrom, USA), absorbent paper mat (Grade 470, American S&S), chloroauric acid (SIGMA), colloidal gold quantitative chromatograph (Skannex, Norway), horizontal centrifuge (Changzhou Bowen) Di company products).

Second, the experimental method

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.

Preparation of colloidal gold-labeled anti-human myoglobin monoclonal antibody: take 10ml of purified water, heat and stir, add 500μl of 10% chloroauric acid solution when the water is boiling, heat and boil for 5 minutes, add 500μl 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. Take a volume of colloidal gold solution 10ml, adjust the pH to 8.3 with 10% potassium carbonate, quickly add 100μg of anti-human myoglobin monoclonal antibody to the final concentration of 10μg/ml, shake the beaker and mix for 30 minutes at room temperature, quickly add 10% 100 ul of bovine serum albumin solution to a final concentration of 1%, while shaking the beaker, standing at room temperature for 30 minutes, centrifuging at 12000 rpm for 20 minutes, carefully aspirating the supernatant; then adding 5 ml of 50 mM phosphate (PBS) buffer, pH 7.4, The pellet was suspended, centrifuged at 12,000 rpm for 20 minutes, and the supernatant was aspirated, and the precipitate was dissolved in 1.0 ml of a phosphate buffer containing 1% of bovine serum albumin and 3% of sucrose, and stored at 4 ° C in the dark.

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 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 gold buffer (1% BSA, 3% sucrose, 50 mM PBS, pH 7.4) to an OD530 of 30, the membrane printer was activated, the antibody was loaded, and a polyester cellulose membrane was taken. The film was started, and the film conditions were set as follows: the moving speed of the airbrush was 30 mm/sec, the liquid pushing speed was 3.0 μl/cm, and the printed film was placed in a dry box, dried at 37 ° C for 6 hours, and then placed in a dry state. The container is stored in a sealed bag.

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 the printing film, set the film conditions: the moving speed of the airbrush is 30mm/sec, and the liquid propelling speed is 0.5μl. /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 chamber to less than 25%, use the colloidal gold-labeled adsorption membrane as the liquid phase-carrying structure, air as the gap structure, and the polyclonal antibody printing film with the nitrocellulose membrane attached A solid phase detecting film, a water absorbent paper film mat as a liquid collecting member, and a PVC sheet as a supporting film. The polyclonal antibody was printed on a film, and a colloidal gold-labeled adsorption film was attached to the PVC film at one end of the nitrocellulose membrane. The colloidal gold-labeled adsorption film was not superimposed on the nitrocellulose membrane, leaving a gap of 1 mm in the nitrocellulose membrane. The other end of the PVC film was pasted with a water-absorbent paper film pad, and the absorbent paper film pad was superimposed with the nitrocellulose film by 1 mm. Place the attached test piece on the slitter and cut into 3.5mm test strips. Put the test strip into the test card, make a test reagent card, put it into the aluminum bag sealed bag with desiccant, seal it on the sealer, and label it. The preparation of the control test reagent card was carried out by using a colloidal gold-labeled adsorption film and a nitrocellulose membrane to be pasted 1 mm, and the others were the same as above.

Detection method: 10 test reagent cards prepared above were taken, and one side of the colloidal gold-labeled adsorption film was placed on the centrifugal turntable in the direction of the proximal end of the centrifuge of the horizontal centrifuge centrifuge, and the adsorption film was labeled with colloidal gold at intervals of 30 seconds. 80ul of different concentrations of human myoglobin solution were added dropwise. After the addition, the reaction was started at 200 rpm for 5 minutes. Then, the test membrane was cleaned by 3,000 rpm for 1 minute, and the test reagent card was taken out. The digital image of the polyclonal antibody blotting strip is read on a colloidal gold quantitative chromatography analyzer (ie, a detector), and image processing is performed to obtain a corresponding chromaticity value. The same test and reaction treatments were also performed on the control test reagent card, and the corresponding chromaticity values were read.

Making a standard curve: taking a known concentration of human myoglobin solution 3.125, 6.25, 12.5, 25, 50, 100 ng / ml human myoglobin solution, using the detection reagent card of the present invention and the control detection reagent card to detect and draw a standard curve . The detection reagent card of the invention adopts a batch detection method. The control test reagent card adopts a single reagent card one-by-one detection method, that is, after a single injection of a single test reagent card, centrifugation is performed to complete the test.

The sample used was 50 ng/ml myoglobin and was prepared in sample dilution buffer.

The experiment was repeated three times and the results were averaged. Statistically calculate the detection values of different test reagent cards.

Third, the experimental results

Card of the present invention is a detection reagent to the colloidal gold assay techniques of this analysis showed a correlation coefficient of the standard sample detection curve invention r ² of 0.982, the correlation coefficient control detection reagent prior art card r ² of 0.966 for the detection of the indicator. The experimental results are shown in Table 14. The test reagent card of the invention performs 10 batch repeatability tests, the average number is 52.07 ng/ml, the standard deviation is 4.27, and the CV value is 8%; and the control test reagent card carries out 10 batches for one batch of repeatability detection. The mean is 69.07 ng/ml, the standard deviation is 12.16, and the CV value is 18%. The detection result of the first sampling of the control reagent card was significantly higher than that of the last sampling test, with a difference of 38 ng/ml, and there was no significant difference in the detection result of the detection reagent card of the present invention. Comparing the two, the method of the invention is obviously superior to the current technology in many aspects such as accuracy, repeatability and convenience of detection.

Table 14 Comparative analysis of the detection results of colloidal gold as an indicator in the present invention (unit: ng/ml)

Example 17, the comparative detection experiment using fluorescein as an indicator in the present invention

First, the experimental materials

Anti-human myoglobin polyclonal antibody (Genagates, USA), anti-human myoglobin monoclonal antibody (Genagates, USA), fluorescent microspheres (fluorescein used as bismuth compound, Shanghai Jieyi Bio), EDC (Pierce products) ), NHS (Pierce products), human myoglobin (Sigma-Aldrich products), fluorescence quantitative analyzer (Shanghai tissue biotechnology company, HG-98), BioFlow printing film instrument (IMAGENE company, USA), Index cutting machine (USA) A-point company), DBF-900 sealing machine (Wenzhou Jiangnan Packing Factory), ACBO dehumidifier (Jiangsu Wuxi Aobo Dehumidifier Company), desktop centrifuge (Eppendoff Company, USA), bovine serum albumin (SIGMA product), nitric acid Cellulose film (AE 99, supplied by Genagates, USA), polyester film (Reemay 2033, product of Alstrom, USA), absorbent paper film pad (Grade 470, American S&S company), horizontal centrifuge (Changzhou) Bowendi products).

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 16.

Fluorescent microsphere labeling: Take 0.5ml fluorescent microspheres, centrifuge 4 times with 0.1M PB of PH6.2, centrifuge at 13000 rpm, reconstitute to 1ml with 0.1M PB pH6.2, add 150ug anti-human myoglobin monoclonal The antibody was mixed, 0.1 M PB of pH 6.2 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.

Preparation of fluorescently labeled antibody adsorption membrane: preparing a polyester cellulose membrane pretreatment liquid containing 0.5% PVA, 50 mM PBS solution, 0.5% BSA, 0.88% NaCl, pH 7.4, and treating the polypolyester cellulose film to be treated The pretreatment liquid was immersed for 1 hour at room temperature, the film was taken out, dried at 37 ° C, and sealed for use. 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 polyester cellulose membrane was taken, the film was printed, and the film conditions were set. The spray gun has a moving speed of 30 mm/sec and a liquid advancing speed of 5.0 μl/cm. The printed film is placed in a dry box, dried at 37 ° C for 6 hours, and then stored in a sealed bag containing a desiccant.

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, start printing the film, set the film conditions: the moving speed of the airbrush is 30mm/sec, the liquid propelling speed is 0.5μl/cm, and it will be printed. The 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 chamber to less than 25%, and use a fluorescently labeled antibody adsorption membrane as a liquid phase bearing structure, air as a gap structure, and a polyclonal antibody printing film with a nitrocellulose membrane attached thereto. A solid phase detecting film, a water absorbent paper film mat as a liquid collecting member, and a PVC sheet as a supporting film. The polyclonal antibody was printed on the membrane, and a fluorescently labeled antibody adsorption membrane was attached to the PVC film on one side of the nitrocellulose membrane. The fluorescently labeled antibody adsorption membrane was not superimposed on the nitrocellulose membrane, leaving a gap of 1 mm in the nitrocellulose. A water-absorbent paper film pad was adhered to the PVC film on the other side of the film, and the absorbent paper film pad was superimposed with the nitrocellulose film by 1 mm. Place the attached test piece on the slitter and cut into 3.5mm test strips. Put the test strip into the test card, make a test reagent card, put it into the aluminum bag sealed bag with desiccant, seal it on the sealing machine, and label it. The preparation of the control reagent card was carried out by using a fluorescently labeled antibody adsorption membrane and a nitrocellulose membrane superimposed with 1 mm, and the others were the same as above.

Detection method: 10 test reagent cards prepared above were taken, and one side of the fluorescent labeling antibody adsorption membrane was placed on the centrifugal rotor in the direction of the proximal end of the centrifuge of the horizontal centrifuge centrifuge, and the fluorescent labeled antibody adsorption membrane was applied at intervals of 30 seconds. 80ul of different concentrations of human myoglobin solution were added dropwise. After the addition, the reaction was started at 200 rpm for 5 minutes. Then, the test membrane was cleaned by 3,000 rpm for 1 minute, and the test reagent card was taken out. The fluorescent value of the polyclonal antibody blotting strip was read on a fluorescence quantitative analyzer (ie, a detector). The same test and reaction treatments were also performed on the control test reagent card, and the corresponding fluorescence values were read.

Making a standard curve: taking a known concentration of human myoglobin solution 3.125, 6.25, 12.5, 25, 50, 100 ng / ml human myoglobin solution, using the detection reagent card of the present invention and the control detection reagent card to detect and draw a standard curve . The detection reagent card of the invention adopts a batch detection method. The control test reagent card adopts a single reagent card one-by-one detection method, that is, after a single injection of a single test reagent card, centrifugation is performed to complete the test.

The experiment was repeated three times and the results were averaged. Statistically calculate the detection values of different test reagent cards.

Third, the experimental results

Card of the present invention is a detection reagent fluorescein measurement result display technique of the invention the correlation coefficient of the standard sample detection curve r ² of 0.991, the correlation coefficient control detection reagent prior art card r ² of 0.978 for the detection of the indicator. The experimental results are shown in Table 15. The test reagent card of the invention performs 10 batch repeatability tests, the average number is 50.87 ng/ml, the standard deviation is 3.44, and the CV value is 7%; and the control test reagent card carries out 10 batches for one batch of repeatability detection. The mean is 70.1 ng/ml, the standard deviation is 13.78, and the CV value is 20%. The detection result of the first sampling of the control reagent card was significantly higher than that of the last sampling test, and the difference was 40 ng/ml, but the detection result of the detection reagent card of the present invention was not significantly different. Comparing the two, the method of the invention is obviously superior to the current technology in many aspects such as accuracy, repeatability and convenience of detection.

Table 15 Comparative analysis of the detection results of colloidal gold as an indicator in the present invention (unit: ng/ml)

Example 18, comparative detection experiment using chemiluminescent indicator in the present invention

First, the experimental materials

Fluorescent microspheres (Shanghai Jieyi Bio), Trehalose (SIGMA), nitrocellulose membrane (Millipore), EDC (PIERCE), NHS (PIERCE), horseradish peroxidase-labeled anti-human myoglobin Cloned antibody (Genagates, USA), anti-human myoglobin polyclonal antibody (Genagates, USA), spectrophotometer (Shanghai Hanhua Scientific Instrument Co., Ltd., 752 UV-Vis spectrophotometer), human myoglobin (Sigma-Aldrich) Products), BioFlow printing film instrument (IMAGENE company, USA), Index cutting machine (A-point company, USA), DBF-900 sealing machine (Wenzhou Jiangnan packaging factory), ACBO dehumidifier (Jiangsu Wuxi Aobo dehumidifier company), Benchtop centrifuge (Eppendoff, USA), bovine serum albumin (abbreviated as BSA, SIGMA), nitrocellulose membrane (AE 99, supplied by Gengates, USA), polyester cellulose membrane (Reemay 2033, Alstrom, USA) Products), absorbent paper film mat (Grade 470, American S&S company), chemiluminescence detector (Promega, Glomax Multi JR Detection System), West Pico luminescent reagent (Thermo scientific), horizontal centrifuge (Changzhou Bowen Digong) Products).

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 16.

Fluorescent microsphere labeling: Take 0.5ml microspheres, centrifuge 4 times with 0.1M phosphate buffer pH6.2, centrifuge at 13000rpm, reconstitute to 1ml with 0.1M phosphate buffer pH6.2, add 2mg horseradish peroxidation. Enzyme-labeled anti-human myoglobin monoclonal antibody, mix, add 250ul of 40mg/ml EDC solution, add 250ul 40mg/ml NHS solution, mix, react at room temperature for 60 minutes, add 20mg of bovine serum albumin, mix Evenly, react at room temperature for 60 minutes. The supernatant was centrifuged, washed 4 times with 0.05 M Tris pH 7.6, reconstituted to 10 ml with 0.5% trehalose, 1% BSA, 0.05 M Tris pH 7.6, and stored at 4 ° C in the dark.

Preparation of fluorescent microsphere-labeled adsorption membrane: The fluorescent microsphere-labeled antibody solution was diluted 3 times with the complex solution, and the other printing method in the preparation of the fluorescent-labeled antibody adsorption membrane was tested.

Preparation of polyclonal antibody printing membrane: Example 16 of the present invention.

Semi-finished product assembly method: start the dehumidifier to reduce the humidity in the operating chamber to less than 25%, use the fluorescent microsphere-labeled adsorption membrane as the liquid phase bearing structure, air as the gap structure, and the polyclonal antibody printing film with the nitrocellulose membrane attached As the solid phase detecting film, the absorbent paper film mat serves as a liquid collecting member, and the PVC sheet serves as a supporting back sheet. The polyclonal antibody was printed on the membrane, and the fluorescent microsphere-labeled adsorption membrane was attached to the PVC film on one side of the nitrocellulose membrane. The fluorescent microsphere-labeled adsorption membrane was not superimposed on the nitrocellulose membrane, leaving a gap of 1 mm in the nitric acid. A water-absorbent paper film pad was stuck on the PVC film on the other side of the cellulose film, and the absorbent paper film pad was superimposed with the nitrocellulose film by 1 mm. Place the attached test piece on the slitter and cut into 3.5mm test strips. Put the test strip into the test card, make a test reagent card, put it into the aluminum bag sealed bag with desiccant, seal it on the sealing machine, and label it. The preparation of the control test reagent card was carried out by using a fluorescent microsphere-labeled adsorption film and a nitrocellulose membrane stacked 1 mm, and the others were the same as above.

Detection method: 10 test reagent cards prepared above were taken, and one side of the fluorescent microsphere-labeled adsorption film was placed on the centrifuge turntable in the direction of the proximal end of the centrifuge of the horizontal centrifuge centrifuge, and the fluorescent microspheres were marked at intervals of 30 seconds. 80ul of different concentrations of human myoglobin solution were added to the adsorption membrane. After the application was completed, the reaction was initiated at 200 rpm for 5 minutes, and the heart was labeled at 1000 rpm for 1 minute, and then labeled with fluorescent microspheres. Add 0.05% Tween-20, 50 mM PBS buffer 80 ul of pH 7.4, 3,000 rpm/separation heart for 30 seconds, wash twice, remove the test strip, and cut the polyclonal antibody film detection line. In a transparent test tube, 200 ul of PBS buffer was added, and ultrasonication was performed for 3 seconds. Take 10ul, add Pico luminescent reagent, put it on the chemiluminescence detector, record the luminescence amount when the reaction is carried out for 2 minutes, the luminescence integration time is 6 seconds, the experiment is repeated three times, the result is averaged, then the concentration-luminescence curve is drawn, and Calculate the correlation coefficient. The same test and reaction treatments were also performed on the control test reagent card, and the corresponding luminescence values were read.

Making a standard curve: taking a known concentration of human myoglobin solution 3.125, 6.25, 12.5, 25, 50, 100 ng / ml human myoglobin solution, using the detection reagent card of the present invention and the control detection reagent card to detect and draw a standard curve . The detection reagent card of the invention adopts a batch detection method. The control test reagent card adopts a single reagent card one-by-one detection method, that is, after a single injection of a single test reagent card, centrifugation is performed to complete the test.

The experiment was repeated three times and the results were averaged. Statistically calculate the detection values of different test reagent cards.

Third, the experimental results

The detection result of the detection reagent card of the present invention using the chemiluminescent enzyme horseradish peroxidase as a luminescent indicator shows that the correlation coefficient r ² of the standard curve sample detection of the present invention is 0.982, and the correlation detection reagent card of the prior art is related. The coefficient r ² is 0.986. The experimental results are shown in Table 16. The test reagent card of the invention performs 10 batch repeatability tests, the average number is 51.06 ng/ml, the standard deviation is 4.16, and the CV value is 8%; and the control test reagent card carries out 10 batch repeatability tests. The mean is 71.52 ng/ml, the standard deviation is 14.37, and the CV value is 20%. The detection result of the first sampling of the control reagent card was significantly higher than that of the last sampling test, and the difference was 42 ng/ml, while the detection result of the detection reagent card of the present invention was not significantly different. Comparing the two, the method of the invention is obviously superior to the current technology in many aspects such as accuracy, repeatability and convenience of detection.

Table 16 Comparative analysis of detection results of luminescent indicators catalyzed by chemiluminescent enzymes in the present invention (unit: ng/ml)

Example 19, the comparative detection experiment of the filter membrane pad as the gap structure of the present invention

First, the experimental materials

Microporous membrane (Shanghai Xingya Purification Material Factory), otherwise the same as in Example 17.

Second, the experimental method

Preparation of human myoglobin solution: same as in Example 16.

Fluorescent microsphere labeling: same as in Example 17.

Preparation of fluorescently labeled antibody adsorption membrane: same as in Example 17.

Polyclonal antibody print preparation: same as in Example 17.

Semi-finished product assembly method: start the dehumidifier to reduce the humidity in the operating chamber to less than 25%, and use a fluorescently labeled antibody adsorption membrane as a liquid phase carrying structure, with a 0.22 μm filter membrane, a 0.45 μm filter membrane and air as a gap structure, The polyclonal antibody printing film of the nitrocellulose membrane serves as a solid phase detecting membrane, the absorbent paper membrane mat serves as a liquid collecting member, and the PVC sheet serves as a supporting backsheet. The polyclonal antibody was printed on the membrane, and a fluorescently labeled antibody adsorption membrane was attached to the PVC film on one side of the nitrocellulose membrane. The fluorescently labeled antibody adsorption membrane was not superimposed on the nitrocellulose membrane, leaving a gap of 1 mm in the nitrocellulose. A water-absorbent paper film pad was adhered to the PVC film on the other side of the film, and the absorbent paper film pad was superimposed with the nitrocellulose film by 1 mm. A filter having a thickness of 1 mm or no filter (air) was attached in the longitudinal direction at the void portion. Place the attached test piece on the slitter and cut into 3.5mm test strips. Put the test strip into the test card, make a test reagent card, put it into the aluminum bag sealed bag with desiccant, seal it on the sealing machine, and label it.

Detection method: 10 test reagent cards prepared above were taken, and one side of the fluorescent labeled antibody adsorption membrane was placed on the centrifugal rotor in the direction of the proximal end of the centrifuge of the horizontal centrifuge centrifuge, and the fluorescent labeled antibody was adsorbed at intervals of 30 seconds. 80ul of different concentrations of human myoglobin solution were added to the membrane. After the sample was added, the reaction was started at 200 rpm for 5 minutes. Then, the membrane was removed by 3,000 rpm for 1 minute, and the detection reagent card was taken out. The fluorescent value of the polyclonal antibody blotting strip was read on a fluorescence quantitative analyzer (ie, a detector).

A standard curve was prepared: the same as in Example 17.

The experiment was repeated three times and the results were averaged. Statistically calculate the detection values of different test reagent cards.

Third, the experimental results

The detection result of the detection reagent card of the present invention using fluorescein as an indicator shows that the correlation coefficient r ² of the detection of the standard curve sample using the 0.22 μm filter mat as the void structure of the present invention is 0.991, and the 0.42 μm filter mat is used as the void structure. standard sample correlation coefficient r ² of the detection curve of 0.979, using air as a detection reagent card void structure of the correlation coefficient r ² of 0.978. The experimental results are shown in Table 17. The detection reagent card of the invention adopts a 0.22 μm filter membrane as a gap structure to perform 10 batch repeatability tests, the average number is 51.28 ng/ml, the standard deviation is 5.18, and the CV value is 10%; the 0.45 μm filter membrane is used as the gap. The structure was subjected to 10 batch repeatability tests with a mean of 51.13 ng/ml, a standard deviation of 4.76, and a CV value of 9%. Ten air was used as the gap structure for repeat detection of one batch, and the mean was 51.07 ng/ml, standard deviation was 4.33, CV value was 8%. There are no obvious differences in the detection results of the three kinds of gap structure detection reagent cards, and they all have many characteristics such as accuracy, repeatability and convenience.

Table 17 Comparative test experiment using filter membrane mat as gap structure in the present invention (unit: ng/ml)

Industrial application

The invention has the following beneficial effects:

1. The liquid phase driven and detected by the centrifugal device is flowed and cleaned on the solid phase detecting membrane, thereby improving the capture and binding ability of the analyte, reducing the background noise interference of the solid phase detecting membrane, improving the detection sensitivity of the method, and realizing the method. High sensitivity detection with existing detection reagents.

2. The present invention uses a centrifugal device to drive the liquid phase detected on the solid phase detection membrane, which changes the current state of the membrane detection method 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 liquid. The uniform flow on the membrane ensures the uniformity of the binding of the analyte on the membrane, which can improve the detection accuracy.

3. Existing membrane detection methods rely on natural flow, and the flow rate of liquid on the membrane slows down with time. It takes more than 15 minutes to complete a test. The invention adopts a centrifugal device to drive the detection liquid phase to flow on the solid phase detecting film, keeps the liquid flowing uniformly on the film, and shortens the detection time quickly.

4. The existing high-sensitivity detection methods adopt multi-step and multi-step drive control, involving the detection and displacement of the sample, the detection phase and the reaction carrier. The invention uses a centrifugal device to drive the liquid phase flow and the sample pump to be injected, and the operation steps are simple.

5. Directly using the indicator mark detection phase on the existing film detection method will generate a strong background signal, and the detection of the object to be inspected cannot be performed. The sealing liquid of the invention can significantly reduce the background signal, and the centrifugal cleaning can effectively remove the residual indicator markers and indicators remaining on the background, thereby effectively improving the detection efficiency and accuracy.

6. 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 method 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 significantly improved, and is applied to detection, analysis and separation. Many areas.

7. The present invention provides a gap structure between the liquid phase bearing structure and the solid phase detecting film to prevent natural flow of the liquid phase on the lateral flow chromatography detecting membrane, which hinders the liquid phase to solid phase detection. Natural flow on the membrane. However, in use, the lateral flow chromatography detection structure is placed on the centrifugal device, and the liquid phase flows through the gap by centrifugal driving, enters the solid phase detection membrane and maintains the flow, thereby initiating the immunodetection reaction. This not only maintains the detection characteristics of the lateral flow chromatography detection device, but also enables batch loading of the liquid phase, initiation of centrifugation, and simultaneous initiation of the detection reaction, effectively improving the accuracy, repeatability and stability of the chromatographic detection. Batch automated testing.

8. The gap structure of the present invention which hinders the natural flow of the liquid phase adopts air or a filter membrane pad, which makes the preparation of the lateral flow detection reagent strip simpler, more convenient and lower in cost, and is advantageous for industrial production of technical products.

9. The operation steps of the invention are simple, easy to realize automatic operation, and at the same time, have the detection method 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 fields.

Claims (14)

1. A centrifugal separation detecting method comprising the steps of: driving a liquid phase in a solid phase detecting membrane by a centrifugal mechanism in a centrifugal separation detecting device to perform immunochromatography;

   The immunochromatography comprises colloidal metal immunochromatography using a colloidal metal as an indicator, fluorescence immunochromatography using fluorescein as an indicator, and chemiluminescent substance and/or chemiluminescent enzyme-mediated luminescence as an indicator of chemiluminescence immunity. Chromatography.
2. A lateral flow chromatography detection reaction initiation control method comprises the steps of: placing a lateral flow chromatography detection mechanism on a centrifugal turntable in the centrifugal separation detecting device, and driving the liquid phase into the solid phase by centrifugal driving Detecting the membrane and maintaining the flow, thereby initiating lateral flow chromatography detection analysis;

   The lateral flow chromatography detecting mechanism comprises a liquid phase bearing structure and a solid phase detecting film disposed on the supporting film, and the liquid phase bearing structure and the solid phase detecting film are left between the solid phase detecting film and the natural flow of the liquid phase. a gap; the liquid phase bearing structure is located at a proximal end of the solid phase detecting membrane.
3. The centrifugal separation detecting method according to claim 1 or the starting control method according to claim 2, wherein the colloidal metal is at least one of colloidal gold, colloidal selenium and colloidal gold magnetic particles;

   The fluorescein is fluorescein isothiocyanate, tetraethyl rhodamine, rhodamine tetramethyl isothiocyanate, phycoerythrin, polydatin chlorophyll protein, propidium iodide, allophytoin and At least one of the hydrazine compounds;

   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 chemiluminescent enzyme is at least one of horseradish peroxidase, alkaline phosphatase, and xanthine oxidase.
4. The centrifugal separation detecting method according to claim 1 or 3, or the starting control method according to claim 2 or 3, wherein the centrifugal separation detecting method or the lateral flow chromatography detecting analysis uses particles as a a carrier carrier for the indicator; the carrier carrier carries the indicator in such a manner that the particle is labeled with the analyte-specific direct conjugate or the analyte-specific indirect conjugate directly labeled with the indicator The microparticles with the indicator directly label the analyte-specific direct conjugate or the analyte-specific indirect conjugate;

   The microparticles are particles capable of forming non-specific binding to proteins and/or the indicator directly and/or by chemical crosslinking and maintaining stability;

   The particle size of the microparticles is 1 nm to 1 um;

   The specific conjugate includes an antigen, an antibody, avidin, biotin or a derivative thereof.
5. The centrifugal separation detecting method according to claim 1, 3 or 4, or the starting control method according to claim 2, 3 or 4, wherein the liquid phase comprises a liquid phase containing a substance to be tested, a liquid phase of the indicator-labeled test substance, a liquid phase of the test substance labeled with the microparticles, a liquid phase of the non-labeled test substance, one of the washing liquid phases, or a combination thereof;

   The test substance is a test substance specific conjugate and a secondary or tertiary specific conjugate thereof;

   The specific conjugate includes an antigen, an antibody, avidin, biotin or a derivative thereof.
6. The centrifugal separation detecting method according to claim 1, 3, 4 or 5 or the starting control method according to claim 2, 3, 12 or 5, wherein the centrifugal separation detecting method further comprises solid phase detecting a step of blocking the membrane blocking solution;

   The blocking solution is a buffer salt solution containing bovine serum albumin and at least one of other soluble protein, skim milk powder, polyethylene glycol, casein, sucrose, surfactant, gelatin, serum, plasma;

   The steps of the closing are as follows:

   1) coating the solid phase detecting membrane with a solid phase detecting membrane coating liquid, wherein the solid phase detecting membrane coating liquid is a solution of an antigen, an antibody, avidin, biotin or a derivative thereof;

   2) washing the solid phase detecting membrane with a washing liquid, which is water, a conventional buffer or a buffer solution containing a surfactant;

   3) immersing the solid phase detecting film with the solid phase detecting film blocking liquid;

   4) washing the solid phase detecting film again with the cleaning liquid;

   5) Dry and spare.
7. The startup control method according to claim 2, 3, 4 or 5, wherein the gap is an air or a filter membrane pad;

   The gap has a width of 0.5 to 3 mm;

   The filter membrane mat has a pore diameter of 0.1 to 5 μm;

   The liquid phase bearing structure is a polypolyester fiber membrane, a glass cellulose membrane, a colloidal gold marker-specific sample pad or a fluorescent marker-specific sample pad.
8. A centrifugal separation detecting device comprising a sampling mechanism and a centrifugal mechanism;

   The centrifugal mechanism includes a centrifugal rotating disc driven by a driving motor and a supporting base; the centrifugal rotating disc is disposed on the supporting base;

   a solid phase detecting film is fixed on the centrifugal rotating disc;

   The injection mechanism includes a liquid phase storage device, a sample tube, and a sample pump, the liquid phase storage device is in communication with the sample tube; the sample pump drives liquid in the liquid phase storage device to enter The sample tube;

   The sample tube loads a liquid in the liquid phase storage device to a proximal end of the solid phase detection membrane.
9. The centrifugal separation detecting device according to claim 8, wherein the solid phase detecting film is disposed along a radial direction of the centrifugal turntable, and is detachably fitted with the centrifugal turntable;

   The two ends of the solid phase detecting membrane are respectively matched with the liquid adsorption dispersing member and the liquid collecting member;

   The liquid adsorption dispersion member is disposed at a proximal end of the centrifugal turntable, and the liquid collection member is disposed at a telecentric end of the centrifugal turntable.
10. The centrifugal separation detecting apparatus according to claim 9, wherein the solid phase detecting film is made of any one of a nitrocellulose membrane, a PVDF membrane, a polyvinylidene fluoride membrane, a nylon membrane, and a DEAE cellulose membrane. ;

    The solid phase detecting film is provided with a backing on one or both sides;

    The liquid adsorption dispersion member is at least one of a colloidal gold labeled adsorption membrane, a fluorescently labeled antibody adsorption membrane, a chemiluminescent label adsorption membrane, and a dispersion membrane;

    The liquid collecting member is a liquid collecting container or made of a water absorbing material.
11. The centrifugal separation detecting device according to claim 9 or 10, wherein the solid phase detecting film is fixed by a solid phase detecting film fixing device;

    The solid phase detecting film fixing device is a snap-like structure, and a sample sampling groove, an observation window and a liquid collecting member outlet are sequentially arranged from one end to the other end;

    After the solid phase detecting film is fixed to the solid phase detecting film fixing device, the spotting groove, the observation window, and the liquid collecting member outlet are sequentially connected to the liquid collecting member, the solid phase detecting film, and The liquid collecting member corresponds.
12. The centrifugal separation detecting device according to claim 9 or 10, wherein the solid phase detecting film is fixed by a solid phase detecting film fixing device;

    The solid phase detecting film fixing device comprises a hard transparent lower cover sheet and a transparent transparent upper cover sheet, and the solid phase detecting film is disposed between the hard transparent lower cover sheet and the transparent transparent upper cover sheet;

    One end or both ends of the solid phase detecting film is a hollow interlayer.
13. The centrifugal separation detecting device according to claim 9 or 10, wherein the centrifugal separation detecting device further comprises an ultrasonic breaking device comprising an ultrasonic generator, a transducer, and a horn which are sequentially connected And ultrasonically disrupting the container;

    The ultrasonic breaking device is disposed at an upper portion of the solid phase detecting film;

    The centrifugation detecting device further includes a detector for detecting a liquid in the solid phase detecting film;

    The detector is one or a combination of a colloidal gold quantitative detector, a fluorescence detector, and a chemiluminescence detector;

    The detection index of the detector is any one of absorbance, fluorescence value, chemiluminescence value, and image digital signal value or a combination thereof.
14. Use of the centrifugal separation detecting device according to any one of claims 8 to 13 in immunodetection.