CN109439517B - Spot hybridization reaction device, application thereof and immunoblotting detection method - Google Patents
Spot hybridization reaction device, application thereof and immunoblotting detection method Download PDFInfo
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Abstract
The invention provides a dot hybridization reaction device, application thereof and an immunoblotting detection method, and relates to the technical field of molecular hybridization experimental devices. The spot hybridization reaction device comprises a sample adding and percolating module and a suction filtration module, wherein the suction filtration module is used for providing a negative pressure environment for the interior of the sample adding and percolating module; the sample adding and percolating module comprises a micropore sample adding plate provided with a sample through hole, a supporting layer provided with a percolating through hole corresponding to the sample through hole and a suction filtration tank body; a first suction unit is arranged in the micropore sample adding plate, and a second suction unit matched with the first suction unit is arranged in the suction filtration tank body; forming a closed inner cavity through adsorption and fixation of the matched suction units; and fixing the hybridization film layer and the support layer under the micropore sample adding plate in sequence. The device is convenient to carry and simple to operate, and can effectively prevent the problem of cross-contamination of the spot hybridization reaction device between samples.
Description
Technical Field
The invention relates to the technical field of molecular hybridization experimental devices, in particular to a dot hybridization reaction device, application thereof and an immunoblotting detection method.
Background
The molecular hybridization technology is a common detection means for biological macromolecules such as nucleic acid, protein and the like, and the nucleic acid molecular hybridization is a method for detecting a specific nucleic acid sequence by using labeled DNA or RNA as a probe; protein molecular hybridization is to use the principle of specific binding of antigen and antibody, and the color developing agent of labeled antibody is used to detect specific polypeptide and protein through chemical reaction.
Spot hybridization is a method of molecular hybridization techniques and generally comprises the steps of: the hybridization membrane is pretreated, then the sample to be detected is spotted on the hybridization membrane, so that the target molecules in the sample are adsorbed on the hybridization membrane, then the molecules marked by the markers, such as antibodies or antigens marked by the markers are used for the target protein molecules, probes marked by the markers are used for the nucleic acid molecules, the nucleic acid molecules are hybridized with the target molecules on the hybridization membrane, and spots are formed on the hybridization membrane after color development.
The conventional spot hybridization technique is time-consuming and labor-consuming to operate, is not easy to standardize, is easy to cause cross contamination between samples, and is not suitable for operation on clinical sites outside a laboratory, so that an improved spot hybridization reaction device is needed in the current market.
In view of this, the present invention has been made.
Disclosure of Invention
The first object of the present invention is to provide a spot hybridization reaction apparatus which alleviates the problems of the prior art, such as the lack of a spot hybridization reaction apparatus which is convenient to carry and easy to operate, and which can effectively prevent cross-contamination between samples.
The second object of the present invention is to provide an application of the spot hybridization reaction apparatus in molecular hybridization experiments, which can be widely applied to molecular hybridization experiments of proteins and nucleic acids.
The third object of the present invention is to provide an immunoblotting detection method using the dot hybridization reaction device, which is simple to operate, has short detection time, has low requirements on detection environment and equipment conditions, and can realize the flow program operation in an integrated reactor.
In order to solve the technical problems, the invention adopts the following technical scheme:
a spot hybridization reaction device comprises a sample adding and percolating module and a suction filtration module for providing a negative pressure environment for the interior of the sample adding and percolating module;
The sample addition and filtration module comprises: the microporous sample adding plate, the hybridization membrane layer, the supporting layer and the suction filtration tank body;
the microporous sample plate is provided with a plurality of sample through holes for reaction, and the supporting layer is provided with percolation through holes corresponding to the sample through holes;
A first suction unit is arranged in the micropore sample adding plate, and a second suction unit matched with the first suction unit is arranged in the suction filtration tank body;
the micropore sample adding plate and the suction filtration tank body are adsorbed and fixed through a first adsorption unit and a second adsorption unit to form a closed inner cavity; and sequentially fixing the hybridization film layer and the supporting layer below the micropore adding plate, wherein the hybridization film layer is in close contact with the lower surface of the micropore adding plate.
Preferably, the microporous template is provided with a rim flange for reserving a space for accommodating at least the hybridization membrane layer and the support layer.
Preferably, the first attraction unit and the second attraction unit respectively and independently comprise one or more of a plurality of magnet units, a plurality of electromagnet units and a plurality of metal units attracted with magnetic substances;
preferably, the first suction unit comprises a plurality of magnet units embedded in the frame flange of the micropore heating plate, and the main body of the suction filtration tank body is made of metal adsorbed with magnetic substances;
preferably, the magnets in the magnet unit are mainly made of magnets with nickel plated surfaces;
preferably, the metal unit adsorbed to the magnetic substance is mainly made of martensitic stainless steel.
Preferably, the sample through holes are arranged in a determinant;
Preferably, the micropore template is provided with 48 sample through holes distributed according to a determinant; the 48 sample through holes are preferably distributed according to 8×6 distribution;
preferably, the micropore template is provided with 96 sample through holes distributed according to a determinant; the 96 sample through holes are preferably distributed according to 8×12 distribution;
Preferably, the volume of the sample through hole is 100-1500 mu L; preferably 500-100. Mu.L; more preferably 600-800. Mu.L.
Preferably, an annular flange along the circumferential direction of the percolation through hole is arranged on the supporting layer, and the annular flange faces the direction of the micropore sample adding plate;
Preferably, a sealing cushion layer is further arranged between the hybridization membrane layer and the supporting layer, and through holes corresponding to the sample through holes are formed in the sealing cushion layer;
preferably, at least one filter paper layer is further arranged between the hybridization film layer and the sealing cushion layer.
Preferably, the cross-sectional area of the groove in the suction filtration tank body decreases with the direction away from the micropore sample adding plate;
preferably, the longitudinal section of the groove in the suction filtration groove body is V-shaped, isosceles trapezoid or semicircular;
preferably, the side wall of the suction filtration tank body is also provided with a suction filtration hole which is used for being communicated with the suction filtration module.
Preferably, the spot hybridization reaction device further comprises a temperature control module;
Preferably, the temperature control module is a constant temperature incubation groove, and a film heating module and a PID temperature control module are embedded in the constant temperature incubation groove.
Preferably, the spot hybridization reaction device further comprises an imaging module;
preferably, the imaging module comprises a camera or a gel imager.
The invention also provides application of the spot hybridization reaction device in a molecular hybridization experiment.
The immunoblotting detection method by using the dot blot reaction device comprises the steps of spotting a sample to be detected into a sample through hole, starting a suction filtration module to suction-filter liquid phase substances in a reaction system into a concave groove of a suction filtration groove body after the sample to be detected reacts with an antibody or antigen marked by a marker, and retaining solid phase substances in the reaction system on a hybridization film to form hybridization spots after color development;
Preferably, the immunoblotting detection method comprises the following steps:
providing a spot hybridization reaction device with a hybridization film layer subjected to activation treatment;
(ii) adding a wet transfer buffer solution into the sample through hole to wet the hybridization membrane layer, and then carrying out suction filtration by using a pressure of 0.03-0.08 Mpa; standing at 20-25deg.C for 20-30min, and vacuum filtering under 0.03-0.05Mpa for 5-10 seconds;
(iii) adding a blocking buffer solution into the sample through hole, incubating for 30-60min at 20-25 ℃, adding an antibody or antigen marked by a marker into the sample through hole after cleaning, and reacting for 40-60min at room temperature under the condition of 35-40 ℃;
(iv) removing the antibody or antigen marked by the marker, incubating for 3-5min at constant temperature by using a chromogenic reagent after washing, filtering for 10 seconds at 0.03-0.05Mpa, adjusting to room temperature, standing for 10min, and taking out the hybridization membrane layer.
Compared with the prior art, the invention has the following beneficial effects:
According to the spot hybridization reaction device provided by the invention, the matched suction units are arranged in the micropore sample adding plate and the suction filtration tank body, so that on one hand, the micropore sample adding plate and the suction filtration tank body are fixed more tightly, the internal space formed between the micropore sample adding plate and the suction filtration tank body is not easy to leak liquid and leak air, the suction filtration effect is ensured, the assembly process is simplified, and the assembly time is saved; on the other hand, the lower surface of the micropore sample adding plate, the hybridization film layer and the supporting layer are tightly adhered, so that samples in the sample through holes are not easy to leak from gaps between the micropore sample adding plate and the hybridization film layer, cross contamination among the samples is avoided, and the detection accuracy is improved. The spot hybridization reaction device provided by the invention can integrally complete the reaction of the sample and form spots on the hybridization film layer, and the reaction place is not required to be replaced in the middle, so that the standardized operation of spot hybridization on the integrated microreactor is realized. The device has small volume, and the suction filtration system can assist the rapid formation of hybridization spots, thereby being convenient for the requirement of rapid detection of experimenters; the spot hybridization reaction device is convenient to carry, simple to operate and quick to detect, can be directly connected with a 220V alternating current power supply or a vehicle-mounted power supply for detection, and has low requirements on detection environment and equipment conditions.
The invention also provides application of the spot hybridization reaction device in a molecular hybridization experiment, which can be widely applied to the molecular hybridization experiment of protein and nucleic acid, and the spot hybridization reaction device has the advantages of convenient carrying, simple operation, rapid detection and lower requirements on detection environment and equipment conditions; can realize the flow program operation in the integrated reactor.
The invention provides an immunoblotting detection method by using the dot hybridization reaction device, which is simple and convenient to operate and low in detection cost, and can effectively avoid cross contamination among samples.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a top view of a microporous template according to example 1 of the present invention;
FIG. 2 is a schematic cross-sectional view of a sample addition and diafiltration module according to example 1 of the present invention;
FIG. 3 is a schematic diagram showing the operation of the apparatus for spot hybridization reaction according to example 1 of the present invention;
FIG. 4 shows the detection of hybridization spots formed by the recombinant protein of pestivirus E2 using the spot hybridization reaction apparatus according to example 1 of the present invention;
FIG. 5 is a standard curve obtained by detecting recombinant protein E2 of swine fever virus according to example 2 of the present invention;
FIG. 6 shows the detection result of the recombinant protein of classical swine fever virus E2 by using a protein electrophoresis quantification method;
FIG. 7 is a standard curve obtained by detecting the recombinant protein of classical swine fever virus E2 using protein electrophoresis quantification method;
FIG. 8 is a standard curve obtained by quantitative detection of the recombinant protein of classical swine fever virus E2 using a double antibody sandwich elisa.
Detailed Description
The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the description of the present invention, it should be noted that, if terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", and the like are used, the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the drawings, only for convenience of describing the present invention and simplifying the description, and does not indicate or imply that the indicated apparatus or element must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and the like, as used herein, are used for descriptive purposes only and are not to be construed as indicating or implying any relative importance.
In the description of the present invention, it should be noted that unless explicitly stated and limited otherwise, the terms "mounted," "connected," and "connected" should be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
The invention provides a spot hybridization reaction device, which comprises a sample adding and percolating module, wherein the sample adding and percolating module mainly comprises a micropore sample adding plate, a hybridization membrane layer, a supporting layer and a suction filtration tank body.
The microporous sample plate is provided with a plurality of sample through holes for reaction, and the support layer is provided with percolation through holes corresponding to the sample through holes, so that liquid in the sample can smoothly flow out through the hybridization membrane during sample suction filtration.
The microporous sampling plate is internally provided with a first suction unit, and the suction filtration tank body is internally provided with a second suction unit matched with the first suction unit. The first suction unit and the second suction unit which are matched with each other have the functions that the contact surfaces of the micropore sample adding plate and the suction filtration tank body are tightly contacted through the adsorption function, and a closed inner cavity is formed after the fixation. The suction unit is used for fixing the micropore sample adding plate and the suction filtration tank body, so that the contact surfaces of the micropore sample adding plate and the suction filtration tank body are closely adhered, no gap is reserved, and leakage of air and liquid when the suction filtration device is used for providing a negative pressure environment for the closed inner cavity is avoided. On the other hand, the hybridization film layer and the supporting layer are sequentially fixed below the micropore adding plate, the hybridization film layer is in close contact with the lower surface of the micropore adding plate, and the hybridization film layer is used as the bottom of the sample through hole so as to be used for containing samples, reagents or reaction systems and avoid liquid outflow. The microporous sampling plate, the hybridization film layer, the supporting layer and the suction filtration tank body can be effectively and tightly integrated into a whole through the adsorption fixation of the first suction unit and the second suction unit.
The spot hybridization reaction device provided by the invention further comprises a suction filtration module for providing a negative pressure environment for the inside of the sample loading and filtration module. When the inside of the sample-adding percolation module is negative pressure, the pressure of the lower surface of the micropore sample-adding plate is lower than the pressure of the upper surface, and a pressure difference is formed between the upper surface and the lower surface of the micropore sample-adding plate, wherein the pressure difference provides a flowing force for liquid phase substances in the sample through holes, so that the liquid phase substances flow into the grooves of the suction-filtration tank body from the sample through holes through the hybridization membrane layer, and the solid phase substances in the sample through holes are retained on the hybridization membrane layer.
When the sample adding device is used, the microporous sample adding plate, the hybridization film layer subjected to pretreatment and the support layer are sequentially stacked and placed, and then the microporous sample adding plate and the suction filtration tank body are tightly adsorbed, so that the microporous sample adding plate, the hybridization film layer, the support layer and the suction filtration tank body are tightly integrated into a whole, namely the sample adding and filtration module. The hybridization film layer is tightly adhered to the lower surface of the micropore sample adding plate under the extrusion action of the supporting layer. Then adding a sample to be detected or a detection reagent into the sample through hole, and providing negative pressure for the inside of the sample adding and percolating module by using the suction filtration module when percolating is required, wherein liquid phase substances in the sample through hole flow into a groove of the suction filtration tank body through the percolating through holes on the hybridization membrane and the supporting layer under the action of negative pressure suction force; and solid phase substances in the through holes of the sample remain on the hybridization membrane to form 'spots'.
According to the spot hybridization reaction device provided by the invention, the matched suction units are arranged in the micropore sample adding plate and the suction filtration tank body, so that on one hand, the micropore sample adding plate and the suction filtration tank body are fixed more tightly, the internal space formed between the micropore sample adding plate and the suction filtration tank body is not easy to leak liquid and leak air, the suction filtration effect is ensured, the assembly process is simplified, and the assembly time is saved; on the other hand, the lower surface of the micropore sample adding plate, the hybridization film layer and the supporting layer are tightly adhered, so that samples in the sample through holes are not easy to leak from gaps between the micropore sample adding plate and the hybridization film layer, cross contamination among the samples is avoided, and the detection accuracy is improved. The spot hybridization reaction device provided by the invention can integrally complete the reaction of the sample and form spots on the hybridization film layer, and the reaction place is not required to be replaced in the middle, so that the standardized operation of spot hybridization on the integrated microreactor is realized. The device has small volume, and the suction filtration system can assist the rapid formation of hybridization spots, thereby being convenient for the requirement of rapid detection of experimenters; the spot hybridization reaction device is convenient to carry, simple to operate and quick to detect, can be directly connected with a 220V alternating current power supply or a vehicle-mounted power supply for detection, and has low requirements on detection environment and equipment conditions. It should be noted that the spot hybridization reaction device provided by the present invention is suitable for being used for example, but not limited to, a protein sample, a DNA sample or an RNA sample, and the present invention is not limited to the type of sample.
In some preferred embodiments, the microporous template and the support layer are each independently made of a high temperature and pressure resistant, transparent or translucent plastic, such as, but not limited to, polypropylene, polycarbonate material. In some alternative embodiments, the material of the hybridization membrane layer is selected according to the kind of biochemical reaction, and the hybridization membrane layer may be, for example, but not limited to, nitrocellulose membrane, cellulose acetate membrane, polyvinylidene fluoride membrane or nylon membrane.
In some preferred embodiments, the frame of the micro-porous template is higher than the surface of the area of the micro-porous template where the sample through holes are distributed, so as to form a frame flange. Optionally, when the frame flange is higher than the surface of the area, on which the sample through holes are distributed, of the micropore sample plate in the direction facing the suction filtration tank body, the frame flange is used for reserving a space for accommodating at least the hybridization film layer and the support layer; optionally, when the frame flange is higher than the area on which the sample through holes are distributed on the micropore sample adding plate in the direction facing away from the suction filtration tank body, the overflowed sample can be prevented from flowing out from the side wall of the micropore sample adding plate during sample application.
In some alternative embodiments, the first engaging unit and the second engaging unit independently include one or more of a plurality of magnet units, a plurality of electromagnet units, and a plurality of metal units that are attracted to a magnetic substance, respectively. For example, but not limited to, the following mating means: the first attraction unit comprises a plurality of magnet units, the second attraction unit comprises a plurality of magnet units, and the magnet units in the first attraction unit and the magnet units in the second attraction unit are opposite in magnetism; the first attraction unit comprises a plurality of magnet units, and the second attraction unit comprises a plurality of metal units attracted with magnetic substances; the first attraction unit comprises a plurality of metal units attracted with magnetic substances, and the second attraction unit comprises a plurality of magnet units; the first attraction unit comprises a plurality of electromagnet units, and the second attraction unit comprises a plurality of metal units attracted with magnetic substances; the first attraction unit comprises a plurality of metal units attracted with the magnetic substance, and the second attraction unit comprises a plurality of electromagnet units. It is understood that the combination of the first suction unit and the second suction unit of the present invention is not limited to the above combinations, as long as the combination can closely adsorb the microporous reaction plate and the suction filtration tank.
In some preferred embodiments, the first engaging unit is embedded in the frame flange of the microporous filler plate, and the second engaging unit is disposed on a surface of the frame flange of the microporous filler plate, which contacts the suction filter tank. In some more preferred embodiments, the first engaging unit includes a plurality of magnet units embedded in the rim flange of the micro-porous sample plate, the magnet units being made of a magnet with a nickel-plated surface; the main body of the suction filtration tank body is made of metal adsorbed by magnetic substances, preferably martensitic stainless steel, namely the whole suction filtration tank body is used as a second suction unit, and the microporous sampling plate is directly adsorbed and fixed on the suction filtration tank body through the frame flange during use, so that the fixation is firmer and no gap is reserved. Wherein, use the Marshall stainless steel as the main part of suction filtration cell body, do benefit to heat conduction, can closely adsorb with the embedded magnet unit of micropore sampling board to equipment, fixed sampling filtration module, high-efficient sealed cross contamination that prevents between the sample mouth.
In some alternative embodiments, the sample through holes on the microporous sample plate are uniformly distributed, for example, but not limited to, distributed according to a determinant or distributed according to a circumferential direction; the sample through holes are preferably arranged in a determinant, and the regular arrangement is convenient for marking and comparing after the samples form spots, for example, the samples can be arranged in an increasing or decreasing way according to the concentration so as to manufacture a standard curve, and the samples and the reference samples are also convenient to be distributed in pairs. In some preferred embodiments, the microwell sample plate is provided with 48 sample through holes or 96 sample through holes to increase throughput of sample detection; the sample through holes on the sample plate with 48 sample through holes are preferably distributed in 8 multiplied by 6; the sample through holes on the sample adding plate provided with 96 sample through holes are preferably distributed in 8 multiplied by 12, and the distribution mode can be compatible with rapid sample application of multiple pipette rows. In some preferred embodiments, the cross-section of the sample through-hole is at least one of circular, regular polygonal, and irregular polygonal. In some preferred embodiments, the volume of the sample through-hole is 100-1500 μl, such as, but not limited to, 100 μl, 200 μl, 500 μl, 600 μl, 800 μl, 1000 μl, 1200 μl or 1500 μl, preferably 500-100 μl; more preferably 600-800. Mu.L; the volume of the sample through holes can be further adjusted and optimized according to the applicable scene of the spot hybridization reaction device, the volume of the sample through holes is smaller, and more sample through holes can be contained on the micropore sample plate so as to increase the detection flux; increasing the volume of the sample through-hole can increase the amount of sample to be measured that is injected at the time of spotting. When the volume of the sample through hole can at least hold 600 mu L of liquid phase substances, the sample to be detected can be further ensured to contain enough solid phase substances which can be fixed on the hybridization film layer through the percolation action, so that the detection sensitivity is improved, and the detection of low-concentration samples and dot blot scanning are facilitated.
In some optional embodiments, the support layer is provided with an annular flange along the circumferential direction of the percolation through hole, the annular flange faces the direction of the micropore sample adding plate, and when the micropore sample adding plate and the suction filtration tank body are closed, the annular flange can tightly squeeze the hybridization film layer on the lower surface of the micropore sample adding plate, so that cross contamination among sample adding holes caused by liquid leakage is prevented, or accurate quantification of samples is prevented due to unclear spot boundaries formed on the hybridization film layer, and therefore, a high-quality blotting result is further ensured. In order to further prevent the liquid in the sample through holes from leaking, in some optional embodiments, a sealing cushion layer is further included between the hybridization film layer and the support layer, the sealing cushion layer is provided with through holes corresponding to the sample through holes, and the sealing cushion layer is preferably made of silicon rubber, fluororubber or polytetrafluoroethylene; in some alternative embodiments, at least one filter paper layer is further disposed between the hybridization membrane layer and the sealing cushion layer.
In some alternative embodiments, the cross-sectional area of the groove in the suction filtration tank body decreases along the direction away from the micropore sample adding plate, so that the liquid phase substances flowing out through suction filtration are collected at the bottom of the groove in the filtration tank body, and the filtrate is conveniently collected. The longitudinal section of the groove in the suction filtration tank body can be, for example, but not limited to, V-shaped, isosceles trapezoid or semicircular.
In some preferred embodiments, the filtering device of the filtering module mainly performs the filtering function, for example, but not limited to, a vacuum pump, a filtering pump or a syringe, preferably a hand-held oilless vacuum pump, a laboratory filtering pump, and a disposable syringe may be optionally used for the filtering operation in veterinary clinics. Preferably, the side wall of the suction filtration tank body is also provided with a suction filtration hole which is communicated with the suction filtration device, and a pipeline can be inserted into the suction filtration hole to be connected with the suction filtration device.
In some alternative embodiments, the dot hybridization reaction apparatus further comprises a temperature control module that provides a controlled isothermal environment for the biochemical reaction. Optionally, the temperature control module may be an electric heater embedded in the suction filtration tank body and/or the microporous reaction plate; alternatively, an external heating module may be selected. Preferably, the external heating module is a constant temperature incubation tank, and a heating module and a regulating and controlling module for controlling the heating module are arranged in the constant temperature incubation tank; the constant temperature incubation groove is also provided with a groove for placing the sample adding percolation module. The heating module is preferably a film heating module, the regulating and controlling module is preferably a PID temperature control module, the film heating module has small volume, and the micro-pore plate constant temperature incubator taking the film heating module as a heating element has small volume, high heating efficiency and convenient use and carrying; the PID temperature control module is a module for regulating and controlling the temperature by using a PID fuzzy control technology, and the PID fuzzy control technology can accurately ensure the temperature control precision, automatically adjust the heating rate and save the waiting time.
In some alternative embodiments, the spot hybridization reaction apparatus further comprises an imaging module to qualitatively or quantitatively record spots on the hybridization membrane layer. Preferably, the imaging module comprises an image acquisition module and an image analysis module. The image acquisition module is used for recording spots on the hybridization film, and the image analysis module is used for assisting relevant patterns and image processing software to read the grey luminosity value of the spots and perform qualitative or quantitative analysis; the imaging module may be, for example but not limited to, a camera or a gel imager, preferably using a gel imager that can be quantitatively analyzed.
The invention also provides application of the spot hybridization reaction device in molecular hybridization experiments, and the application and the spot hybridization reaction device are based on the same inventive concept, so that the spot hybridization reaction device has all the beneficial effects of the spot hybridization reaction device and is not repeated herein. It should be noted that, the application of the present invention in the molecular hybridization experiment only refers to the detection of whether the sample to be detected contains the target substance by using the molecular hybridization technology, and is not used for diagnosis and treatment of diseases.
The application of the spot hybridization reaction device in the molecular hybridization experiment can be applied to detection of protein samples, such as detection of antigens and antibodies of livestock and poultry blood samples in veterinary clinic, and rapid, accurate, qualitative and quantitative detection of effective antigens in the research and development and production processes of veterinary vaccines, and simultaneous detection of various antibodies and antigens, thus realizing on-site real-time evaluation of vaccine antigen content, and the detection time only needs 90-120min; the method can also be applied to detection of nucleic acid samples, such as detection of DNA and/or RNA in livestock and poultry blood samples, tissue samples and cell samples in veterinary clinic; the spot hybridization reaction device provided by the invention is used for molecular hybridization experiments, is simple and convenient to operate and low in detection cost, and can effectively avoid cross contamination among samples. The above-mentioned purpose of detecting DNA and/or RNA in a blood sample, a tissue sample, and a cell sample of livestock and poultry in veterinary clinic is merely to detect whether a target molecule is contained, and does not mean that the detection result can be directly used for diagnosis and treatment of diseases.
The invention also provides an immunoblotting detection method by the dot hybridization reaction device, which adopts the flow program operation in the integrated reactor, and is simple and convenient to operate, and the method comprises the following steps: and spotting a sample to be detected into the sample through hole, starting a suction filtration module to suction-filter liquid phase substances in a reaction system into a groove of a suction filtration groove body after the sample to be detected reacts with the antibody or antigen marked by the marker, and forming hybridization spots after color development, wherein the solid phase substances in the reaction system are retained in the hybridization film.
The immunoblotting detection method is implemented in the following way, and the effect is better:
hybridization membrane preparation and pretreatment: the hybridization film layer is cut into a size matched with the lower surface of the micropore reaction plate, methanol is used for activating for 10s, the hybridization film layer is soaked in wet transfer buffer solution, and a thin filter paper pad with the same size is additionally prepared below the sealing cushion layer to prevent leakage. And then combining all components of the sample-adding and infiltration module, ensuring that the hybridization film layer is tightly attached to the lower surface of the microporous reaction plate, taking care of avoiding bubbles from generating between films, completing the assembly and the fixation of the sample-adding and infiltration module through a first suction unit in the microporous sample-adding plate and a second suction unit in the suction filtration tank body, and connecting the sample-adding and infiltration module and the suction filtration module through pipelines.
(Ii) membrane hydration and spotting: adding wet rotation buffer solution into the sample through hole to wet the hybridization film layer, starting the suction filtration module, performing suction filtration by using the pressure of 0.03-0.08Mpa, and rapidly closing the suction filtration module when visible liquid flows out from the bottom of the hole; adding a sample to be detected into the sample through hole, recording the sample adding sequence, wherein the sample adding volume is preferably 100-600 mu L/hole, the reaction temperature is 20-25 ℃, standing for 20-30min, enabling the liquid to automatically flow out under the action of gravity, and then carrying out suction filtration for 5-10 seconds by using 0.03-0.05Mpa pressure; the microplate centrifugation equipment can be selected to centrifuge for 5min under the condition of 3000-4000rpm to finish quick loading.
(Iii) blocking and incubating: adding a blocking reagent into each hole, and incubating for 30-60min at 20-25 ℃; washing 3-5 times with PBST, 100-3000 mu L/well; 100-200 mu L of a label-labeled antibody or antigen, preferably horseradish peroxidase or colloidal gold, is added to each well, and the temperature of the incubator is adjusted to 37 ℃ and the reaction is carried out for 40-60min at room temperature.
(Iv) developing and imaging: after aspiration of the labeled antibody or antigen, the wells were washed 3-5 times with PBST, 100-300. Mu.L/well. Adding developer for development, preferably ECL chemiluminescent agent, DAB, etc., incubating at constant temperature for 3-5min, suction filtering at 0.03-0.05Mpa for 10s, adjusting to room temperature, standing for 10min, disassembling the sample-adding percolation module, taking out hybridized film, photographing with gel imaging system, collecting signal, and analyzing data.
The advantageous effects of the present invention are further described below in connection with preferred embodiments.
Example 1
This example provides a dot hybridization reaction apparatus, as shown with reference to FIGS. 1, 2 and 3. The spot hybridization reaction apparatus provided in this embodiment includes a sample-adding percolation module 100, and the sample-adding percolation module 100 includes: the sample adding plate 110 is provided with a sample through hole 111 and a frame flange 112, the sample adding and percolating module 100 further comprises a hybridization membrane layer 120, a filter paper layer 130, a sealing cushion layer 140 and a supporting layer 150, and the supporting layer 150 is provided with an annular flange 151; the sample-adding and percolating module 100 further comprises a suction filtration tank 160, wherein a groove 161 is arranged on the suction filtration tank 160, and suction filtration holes 162 are arranged on the side wall of the suction filtration tank 160;
the spot hybridization reaction device provided in this embodiment further includes a suction filtration module: the oil-free vacuum pump comprises an oil-free vacuum pump 210, a pressure gauge 220 and a suction filtration pipeline 230, wherein a suction filtration switch 231 is arranged on the suction filtration pipeline 230;
The spot hybridization reaction apparatus provided in this embodiment further includes a constant temperature incubation tank 300: the side wall of the constant temperature incubation groove 300 is provided with a liquid crystal control panel 310.
The microwell sample plate 110 is provided with sample through holes 111 distributed according to 8×12 determinant, the cross-sectional area of the sample through holes 111 is circular, and the volume of the sample through holes 111 is 600 μl; the raised portion of the rim flange 112 of the microporous filler plate 110 facing the suction filter bowl 160 serves as a stop to reserve space for the hybridization membrane layer 120, the filter paper layer 130, the sealing cushion layer 140, and the support layer 150, and the raised portion facing away from the suction filter bowl 160 serves to prevent liquid from escaping from the microporous filler plate 110.
The rim flange 112 of the micro-hole template 110 is 1.5cm in height, the sample through hole 111 is 0.5cm in height, the dimensions of the rest of the micro-hole template 110 are shown in table 1, the dimension marks are shown in fig. 1, wherein a is the width of the micro-hole template 110; b is the distance from the center of the sample through holes 111 in the 1 st row to the center of the sample through holes 111 in the same column in the 8 th row; c is the length of the micropore template 110; d is the distance from the center of the sample through holes 111 in column 1 to the center of the sample through holes 111 in the same row in column 12; e is the distance between the centers of two adjacent rows or columns of sample through holes 111; f is the diameter of the sample through hole 111.
Table 1 dimensional parameters of microporous template 110
| Units: mm (mm) | A | B | C | D | E | F | G |
| 96-Well plate | 85.5 | 63 | 126 | 99 | 9 | 8 | 5 |
The first attraction unit is a nickel-plated powerful magnet which is embedded in the shell of the frame flange 112 and is filled with the whole frame flange 112, so that the magnet surrounds the area of the micropore template 110, in which the sample through holes 111 are distributed, to form a loop; the second suction unit is the suction filtration tank 160 itself made of martensitic stainless steel. The hybridization film layer 120, the filter paper layer 130, the sealing cushion layer 140 and the supporting layer 150 are sequentially laminated below the microporous sample adding plate 110 after the microporous sample adding plate 110 and the suction filtration tank 160 are adsorbed and fixed, and the hybridization film layer 120 is tightly adhered to the lower surface of the microporous sample adding plate 110, so that the hybridization film layer 120 is used as the bottom of the sample through hole 111 for containing samples, reagents or reaction systems; a filter paper layer 130 is arranged below the hybridization film layer 120, a sealing cushion layer 140 is arranged below the filter paper layer 130, and a through hole corresponding to the sample through hole 111 is arranged on the sealing cushion layer 140; the support layer 150 is arranged below the sealing cushion layer 140, the support layer 150 is provided with an annular flange 151 corresponding to the sample through hole 111 and along the circumferential direction of the infiltration through hole, and the annular flange 151 protrudes towards the direction of the microporous reaction plate so as to further enhance the extrusion effect on the hybridization film layer 120, so that the hybridization film layer 120 is tightly abutted against the lower surface of the microporous template 110. The housing and the support layer 150 of the micro-porous template 110 of the present embodiment are made of polypropylene; the hybrid film layer 120 uses a polyvinylidene fluoride film (PVDF film); the sealing cushion layer 140 is made of silicone rubber.
As shown in fig. 2, a longitudinal section of a groove 161 in the suction filtration tank 160 is V-shaped, a suction filtration hole 162 is provided on a side wall of the suction filtration tank, a suction filtration pipeline 230 can be inserted into the suction filtration hole 162 to be connected with an oil-free vacuum pump 210, a suction filtration switch 231 is provided on the suction filtration pipeline 230, a pressure gauge 220 for monitoring pressure is further provided on the oil-free vacuum pump 210, and the suction filtration pipeline 230 used in this embodiment is a silicone tube.
The groove on the constant temperature incubation groove 300 of the embodiment is identical to the sample-adding percolation module 100, the constant temperature incubation groove 300 uses a thin film heating module as a heating element, the temperature is controlled by a PID temperature control module, and a liquid crystal control panel 310 is further arranged on the side wall of the constant temperature incubation groove 300 to adjust and display the reaction temperature and time.
When in use, the suction filtration tank body 160, the supporting layer 150, the sealing cushion layer 140, the filter paper layer 130, the hybridization film layer 120 and the micropore sample adding plate 110 are arranged from bottom to top in sequence, and the suction filtration tank body 160 and the micropore sample adding plate 110 are adsorbed and fixed to form a whole, namely the sample adding percolation module 100. The hybridization film layer 120 is tightly adhered to the lower surface of the microporous heating plate 110 under the pressing action of the support layer 150. The load lock 100 is then placed in a constant temperature incubation tank 300 and the load lock 100 is connected to an oil free vacuum pump 210 by a suction line 230. Then adding a sample to be detected or a reagent for detection into the sample through hole 111, regulating the constant temperature incubation tank 300 to a required reaction temperature, starting the oilless vacuum pump 210 to provide negative pressure for the inside of the sample-adding percolation module 100 when percolation is required, and enabling liquid phase substances in the sample through hole 111 to flow into the groove 161 of the suction-filtration tank body 160 through the hybridization membrane, the through hole on the sealing cushion layer 140 and the percolation through hole on the supporting layer 150 in sequence under the action of negative pressure suction force; at the same time, the solid phase substance in the sample through hole 111 stays on the hybridization membrane, and forms a "spot" after color development. After removal of the hybridization membrane layer 120, the signals are photographed using a gel imaging system and analyzed.
Example 2
In this example, the spot hybridization reaction apparatus provided in example 1 was used to measure the content of E2 recombinant protein in the production process of the E2 recombinant baculovirus inactivated vaccine of swine fever virus. The method comprises the following steps:
Hybridization membrane layer 120 preparation and pretreatment: the cut 10.5cm×7.5cm PVDF film was used as the hybridization film layer 120, activated with methanol for 10s, immersed in wet buffer, and a thin filter paper pad of the same size was prepared under the sealing pad layer 140 to prevent leakage.
(Ii) assembling the load diafiltration module 100: the filtration tank 160, the supporting layer 150, the sealing gasket layer 140, the filter paper layer 130, the hybridization membrane layer 120 and the microporous template 110 are arranged from bottom to top in sequence, and the components are fixed and then placed in the constant temperature incubation tank 300, and the oil-free vacuum pump 210 is connected by using a silica gel tube.
(Iii) film hydration and spotting: 200 mu L of wet buffer solution is added into the sample through hole 111 of the micropore sample plate 110 to wet the PVDF membrane, the PVDF membrane is filtered by an oil-free vacuum pump 210 under the pressure of 0.05Mpa, and when the liquid flows out from the sample through hole 111, the oil-free vacuum pump 210 is rapidly closed; e2 recombinant protein to be detected and a standard substance (recombinant E2 protein diluted by 2-time gradient, wherein the concentration is 250, 125, 62.5, 31.25 and 15.625 mu g/ml) are sequentially added into a sample through hole 111 by a pipetting gun, repeated control is arranged, 200 mu L/hole is arranged, a constant temperature incubation groove is regulated to 300-25 ℃, standing is carried out for 30min, so that liquid automatically flows out under the action of gravity, and then suction filtration is carried out for 5 seconds by using 0.03-0.05Mpa pressure.
(Iv) blocking and incubating: 200 mu L of PBST buffer containing 5% skimmed milk powder is added to each well, and incubated at 25 ℃ for 30min; wash 3 times with PBST, 200 μl/well; 100. Mu.L of peppery root peroxidase-labeled antibody was added to each well, and the incubation bath was adjusted to 300℃to 37℃and reacted for 60 minutes.
(V) step five, developing and imaging: after aspirating the enzyme-labeled antibody with a pipette, the cells were washed 3 times with PBST wash, 200. Mu.L/well. And (3) adding an ECL chemiluminescent agent for development, incubating for 3min at constant temperature, performing suction filtration for 10 seconds at 0.03Mpa, adjusting to room temperature, standing for 10min, disassembling the sample-adding percolation module 100, taking out the PVDF membrane, photographing by using a UV gel imaging system, collecting signals, analyzing data, constructing a standard curve, and calculating the concentration of the E2 recombinant protein to be detected.
Hybridization spots on hybridization membrane layer 120 are shown in FIG. 4; the standard curve is shown in fig. 5, where y=0.0006 x-18.6798; r 2 = 0.9971.
Effect example
The samples and the standard detected in example 2 were quantified by a protein electrophoresis gray value quantification method and a double antibody sandwich elisa method, respectively, and the accuracy of the three quantification methods was compared. The results of the protein electrophoresis quantification method are shown in fig. 6, and the standard curve is shown in fig. 7, wherein y= 45.594x-2.6827; r 2 =0.998. The standard curve constructed by the double antibody sandwich elisa assay is shown in fig. 8, where y=0.0003 x-3.4359; r 2 =0.9985. The results of the three methods are shown in table 2:
table 2 comparison of the results of the detection method, the quantitative method for the gray-scale spectrophotometry of the protein electrophoresis and the quantitative detection of the recombinant E2 protein by the double-antibody sandwich elisa method provided in example 2
The results show that the difference among the quantitative results of the recombinant E2 protein obtained by the three methods is not obvious, the fitting degree of the constructed standard curve is not lower than 0.99, and the deviation of the measurement results among repeated samples is less than 5%. In terms of detection accuracy, the detection sensitivity and accuracy of the double-antibody sandwich elisa method are highest, the spot hybridization method is next, and the protein electrophoresis method is next; the detection cost is elisa highest, and the spot hybridization is lowest; in terms of the difficulty and the detection time of the detection method, the spot hybridization is the simplest, the detection period is the shortest, and the high-flux detection can be realized as elisa.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (21)
1. The spot hybridization reaction device is characterized by comprising a sample adding and percolating module and a suction filtration module for providing a negative pressure environment for the interior of the sample adding and percolating module;
The sample addition and filtration module comprises: the microporous sample adding plate, the hybridization membrane layer, the supporting layer and the suction filtration tank body;
the microporous sample plate is provided with a plurality of sample through holes for reaction, and the supporting layer is provided with percolation through holes corresponding to the sample through holes; an annular flange along the circumferential direction of the percolation hole is arranged on the supporting layer, and faces the direction of the micropore sample adding plate;
The micropore sample adding plate is provided with a frame flange for reserving a space, and the space is used for accommodating at least the hybridization film layer and the supporting layer;
A first suction unit is arranged in the micropore sample adding plate, and a second suction unit matched with the first suction unit is arranged in the suction filtration tank body;
the first suction unit comprises a plurality of magnet units embedded in the frame flange of the micropore sample adding plate, and the main body of the suction filtration tank body is a second suction unit which is made of metal adsorbed with magnetic substances;
The micropore sample adding plate and the suction filtration tank body are adsorbed and fixed by a first suction unit and a second suction unit to form a closed inner cavity; and sequentially fixing the hybridization film layer and the supporting layer below the micropore adding plate, wherein the hybridization film layer is in close contact with the lower surface of the micropore adding plate.
2. The spot hybridization reaction apparatus according to claim 1, wherein the magnet in the magnet unit is mainly made of a magnet whose surface is plated with nickel.
3. The spot hybridization reaction apparatus according to claim 1, wherein the metal unit adsorbed to the magnetic substance is mainly made of martensitic stainless steel.
4. The spot hybridization reaction device according to claim 1, wherein the sample through holes are arranged in a matrix.
5. The spot hybridization reaction apparatus according to claim 4, wherein the microwell template has 48 sample through holes distributed according to a determinant; the 48 sample through holes are distributed according to 8×6.
6. The spot hybridization reaction apparatus according to claim 4, wherein the microwell template has 96 sample through holes distributed according to a determinant; the 96 sample through holes are distributed according to 8×12.
7. The spot hybridization reaction apparatus according to claim 1, wherein the volume of the sample through hole is 100 to 1500. Mu.L.
8. The spot hybridization reaction apparatus according to claim 7, wherein the volume of the sample through hole is 500 to 100. Mu.L.
9. The spot hybridization reaction apparatus according to claim 7, wherein the volume of the sample through hole is 600 to 800. Mu.L.
10. The spot hybridization reaction apparatus according to claim 1, further comprising a sealing cushion layer between the hybridization membrane layer and the support layer, wherein a through hole corresponding to the sample through hole is provided on the sealing cushion layer.
11. The spot hybridization reaction device according to claim 10, wherein at least one filter paper layer is further provided between the hybridization membrane layer and the sealing cushion layer.
12. The spot hybridization reaction apparatus according to claim 1, wherein the cross-sectional area of the groove in the suction filtration tank decreases in a direction away from the microporous sampling plate.
13. The spot hybridization reaction apparatus according to claim 12, wherein the longitudinal cross section of the groove in the suction filtration tank is V-shaped, isosceles trapezoid or semicircular.
14. The spot hybridization reaction apparatus according to claim 1, wherein the side wall of the suction filtration tank is further provided with a suction filtration hole for communicating with a suction filtration module.
15. The apparatus of claim 1, further comprising a temperature control module.
16. The spot hybridization reaction apparatus according to claim 15, wherein the temperature control module is a constant temperature incubation tank with a thin film heating module and a PID temperature control module embedded therein.
17. The spot hybridization reaction device according to claim 1, further comprising an imaging module.
18. The spot hybridization reaction apparatus according to claim 17, wherein the imaging module comprises a camera or a gel imager.
19. Use of the spot hybridization reaction apparatus according to any one of claims 1 to 18 in a molecular hybridization assay.
20. An immunoblotting detection method using the dot blot hybridization reaction device according to any of claims 1-18, characterized by comprising the steps of spotting a sample to be detected into the sample through hole, starting a suction filtration module to suction-filter liquid phase substances in a reaction system into grooves of a suction filtration tank body after the sample to be detected reacts with an antibody or antigen marked by a marker, retaining solid phase substances in the reaction system on a hybridization film, and forming hybridization spots after color development.
21. The immunoblot detection method according to claim 20, characterized in that it comprises the steps of:
providing a spot hybridization reaction device with a hybridization film layer subjected to activation treatment;
(ii) adding a wet transfer buffer solution into the sample through hole to wet the hybridization membrane layer, and then carrying out suction filtration by using a pressure of 0.03-0.08 Mpa; standing at 20-25deg.C for 20-30min, and vacuum filtering under 0.03-0.05Mpa for 5-10 seconds;
(iii) adding a blocking buffer solution into the sample through hole, incubating for 30-60min at 20-25 ℃, adding an antibody or antigen marked by a marker into the sample through hole after cleaning, and reacting for 40-60min at room temperature under the condition of 35-40 ℃;
(iv) removing the antibody or antigen marked by the marker, washing, incubating for 3-5min at constant temperature by using a chromogenic reagent, filtering for 10 seconds at 0.03-0.05Mpa, adjusting to room temperature, standing for 10min, and taking out the hybridization membrane layer.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811604174.3A CN109439517B (en) | 2018-12-26 | Spot hybridization reaction device, application thereof and immunoblotting detection method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811604174.3A CN109439517B (en) | 2018-12-26 | Spot hybridization reaction device, application thereof and immunoblotting detection method |
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| CN109439517B true CN109439517B (en) | 2024-07-05 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5217592A (en) * | 1985-09-26 | 1993-06-08 | Jones Kenneth W | Electrophoresis and vacuum molecular transfer apparatus |
| JPH0751099A (en) * | 1993-08-11 | 1995-02-28 | Toyobo Co Ltd | Method for examining sequence of nucleic acid and examination apparatus |
| CN209352895U (en) * | 2018-12-26 | 2019-09-06 | 天康生物股份有限公司 | Dot hybridization reaction unit and molecule hybrid experiment system |
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5217592A (en) * | 1985-09-26 | 1993-06-08 | Jones Kenneth W | Electrophoresis and vacuum molecular transfer apparatus |
| JPH0751099A (en) * | 1993-08-11 | 1995-02-28 | Toyobo Co Ltd | Method for examining sequence of nucleic acid and examination apparatus |
| CN209352895U (en) * | 2018-12-26 | 2019-09-06 | 天康生物股份有限公司 | Dot hybridization reaction unit and molecule hybrid experiment system |
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