[go: up one dir, main page]

WO2018176262A1 - Système de détection automatique pour immunotransfert - Google Patents

Système de détection automatique pour immunotransfert Download PDF

Info

Publication number
WO2018176262A1
WO2018176262A1 PCT/CN2017/078576 CN2017078576W WO2018176262A1 WO 2018176262 A1 WO2018176262 A1 WO 2018176262A1 CN 2017078576 W CN2017078576 W CN 2017078576W WO 2018176262 A1 WO2018176262 A1 WO 2018176262A1
Authority
WO
WIPO (PCT)
Prior art keywords
strip
liquid
film
reaction
shaped hydrophilic
Prior art date
Application number
PCT/CN2017/078576
Other languages
English (en)
Chinese (zh)
Inventor
黄世海
Original Assignee
武汉优视科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201710190190.1A external-priority patent/CN107045067B/zh
Priority claimed from CN201710189295.5A external-priority patent/CN106932596A/zh
Priority claimed from CN201710190186.5A external-priority patent/CN107045066A/zh
Priority claimed from CN201710189318.2A external-priority patent/CN106680514B/zh
Priority claimed from CN201710188888.XA external-priority patent/CN106814199B/zh
Application filed by 武汉优视科技有限公司 filed Critical 武汉优视科技有限公司
Publication of WO2018176262A1 publication Critical patent/WO2018176262A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices

Definitions

  • the invention belongs to the field of protein analysis, and more particularly to an automatic detection system for immunoblotting.
  • Western Blot is a technique in which proteins are transferred to a membrane and then detected using antibodies. Immunoblots are commonly used to identify certain proteins and to perform qualitative and semi-quantitative analysis of proteins. Combined with chemiluminescence detection, it is possible to simultaneously compare the difference in expression levels of the same proteins in multiple samples. Since immunoblotting has high resolution of SDS-PAGE and high specificity and sensitivity of solid phase immunoassays, it has become a conventional technique for protein analysis.
  • immunoblot analyzers In order to achieve highly efficient automated analysis, a wide variety of immunoblot analyzers are available (patent document CN103616526A). However, the above immunoblot analyzer usually performs parallel detection of a plurality of samples to be tested by using a plurality of reagent bottles or reaction tanks, and simultaneously shakes the reagent bottles with a shaker to accelerate the reaction speed.
  • the present invention provides an automatic detection system for immunoblotting, which aims to simplify the structure of the automatic detection system while achieving in-situ detection.
  • an automatic detection system for an immunoblot comprising a reaction device, a dosing device, and a mechanical motion device;
  • the surface of the reaction device has a plurality of parallel strip-shaped hydrophilic regions spaced by a hydrophobic region, each strip-shaped hydrophilic region having one or more bonding regions along a length thereof, and the liquid adding device has a strip a plurality of liquid addition channels corresponding to the hydrophilic regions, wherein the control end of the mechanical movement device is connected to the reaction device or the liquid addition device;
  • Each of the strip-shaped hydrophilic regions is used to bind different test solutions, thereby independently performing immunoblot detection of the target antibody in different test solutions, and the binding region has a target antigen for use in Detecting binding of the target antibody in the solution;
  • the liquid adding device is for adding a reaction liquid to the strip-shaped hydrophilic region, and the mechanical motion device is for controlling the relative movement of the water outlet of the liquid addition channel along the strip-shaped hydrophilic region to promote The relative movement of the reaction zone with the binding zone of the strip-shaped hydrophilic region;
  • the reaction solution comprises a solution to be tested, a buffer solution, a secondary antibody solution or a color developing solution.
  • the reaction device comprises a substrate and an immunoblot film covering the surface of the substrate, the immunoblot film having a plurality of parallel strip-shaped hydrophilic regions spaced by a hydrophobic region.
  • the immunoblotting film is attached to the surface of the substrate.
  • the bottom surface of the immunoblotting film and the upper surface of the substrate have a magnetic coating layer, and the immunoblotting film is magnetically adsorbed on the upper surface of the substrate.
  • the upper surface of the substrate is provided with a buckle that fixes the immunoblotting film to the upper surface of the substrate.
  • the substrate is in the form of a flat plate.
  • the mechanical motion device is a linear motor disposed on both sides of the immunoblotting film, and the control end of the linear motor is connected with a liquid adding device for controlling the reciprocating channel to reciprocate along the direction of the strip-shaped hydrophilic region. motion.
  • both sides of the automatic detector have slide rails parallel to the strip-shaped hydrophilic regions.
  • the substrate has a circular axis shape, and the strip-shaped hydrophilic region is perpendicular to a central axis of the circular-axis substrate.
  • the cylindrical substrate has a diameter of 2 cm to 6 cm and a width of 2 cm to 6 cm.
  • the mechanical motion device is a rotary electric machine, and a control end of the rotary electric machine is coupled to the circular shaft-shaped substrate to control the rotation of the immunoblood film around the central axis of the substrate.
  • the distance between the water outlet of the liquid addition channel and the corresponding strip-shaped hydrophilic region is 0.1 mm to 2 mm.
  • the liquid adding device comprises a liquid adding tube and a liquid adding pump, wherein the liquid adding tube and the liquid adding pump are in communication to form a plurality of liquid adding channels corresponding to the strip-shaped hydrophilic regions, the adding liquid
  • the water inlet of the pump serves as a water inlet of the liquid adding device
  • the water outlet of the liquid adding pipe serves as a water outlet of the liquid adding device.
  • the automatic detection system further includes a waste liquid collection device for collecting excess reaction liquid.
  • the waste liquid collecting device includes a plurality of liquid collecting tubes and a vacuum pump; the water inlets of the plurality of liquid collecting tubes are respectively opposed to the plurality of strip-shaped hydrophilic regions as the waste liquid collecting device
  • the water inlet is connected to a vacuum pump for collecting excess reaction liquid on the surface of the strip-shaped hydrophilic region, and the vacuum pump is used to pump the reaction liquid.
  • the waste liquid collection device includes a plurality of drainage tubes and a sump; the water inlets of the plurality of drainage tubes respectively connect a plurality of strip-shaped hydrophilic regions to serve as the waste collection device a water sump, the sump is disposed below a water outlet of the plurality of drainage tubes, and the drainage tube is configured to take out excess reaction liquid, The sump is used to collect excess reaction liquid.
  • the automatic detection system further comprises a drying device.
  • the drying device is a fan or an exhaust fan having an air volume of 2 m 3 /min to 5 m 3 /min.
  • the automatic detection system further comprises an ultrasound probe disposed opposite the strip-shaped hydrophilic region for accelerating the reaction speed of the immunoblotting.
  • the automatic detection system further comprises a signal analysis device, the signal analysis device is disposed opposite to the surface of the reaction device, and the signal analysis device is configured to acquire a detection signal of the immunoblot.
  • the signal analysis device comprises a light source and an image sensor; the light source is for emitting light such that the strip-shaped hydrophilic region forms a detection signal of an immunoblot, and the image sensor is used to acquire a detection signal of the immunoblot.
  • the light source is a visible light source or a laser light source, and the visible light source has an emission wavelength of 490 nm to 570 nm.
  • the signal analysis device further comprises a data processor, an output of the image sensor is connected to an input end of the data processor, and the data processor is configured to obtain an immunoblot according to the image detection signal Test results.
  • the automatic detection system further includes an alarm device, an output of the signal analysis device is connected to an input end of the alarm device, and the alarm device is configured to acquire an end alarm of the immunoblot according to the detection result. Or failure alert.
  • the automatic detection system further comprises a control liquid for adding to the strip-shaped hydrophilic region corresponding to the liquid addition channel by any one of the liquid addition channels, and combining with the strip-shaped hydrophilic region The region reacts to indicate the end of the immunoblotting reaction or the reaction fails.
  • an immunoblotted film having a plurality of parallel strip-shaped hydrophilic regions spaced apart by a hydrophobic region, the strip-shaped hydrophilic region having one or more a binding region having a target antigen for binding to an immunoblotted antibody of interest.
  • the strip-shaped hydrophilic region has a plurality of binding regions, the plurality of binding regions including an experimental region and a control region.
  • control zone comprises a positive control zone, a negative control zone or a conjugate control zone.
  • the material of the strip-shaped hydrophilic region is nitrocellulose, nylon or polyvinylidene fluoride.
  • the hydrophobic region has a silane group.
  • the material of the hydrophobic region is polydimethylsiloxane or a derivative thereof.
  • the strip-shaped hydrophilic region and the hydrophobic region have a width of 1 mm to 5 mm.
  • the immunoblotting film is wound into a reel structure in the direction of the strip-shaped hydrophilic region.
  • the reel structure has a diameter of 2 cm to 6 cm and a width of 2 cm to 6 cm.
  • the reel structure is wound on a circular shaft-shaped substrate.
  • the material of the hydrophobic membrane being nitrocellulose, nylon or polyvinylidene fluoride;
  • a hydrophobic coating is used to form a hydrophobic region on the hydrophobic membrane to separate the hydrophobic membrane into a plurality of strip-shaped hydrophilic regions while separating the binding bands into binding regions such that each strip-like hydrophilic region has the same number of target antigens Combination zone.
  • the surface of the hydrophobic film has a groove that cooperates with the strip-shaped hydrophilic film for fixing the strip-shaped hydrophilic film.
  • a liquid adding device for the above automatic detector, the liquid adding device having one or more water inlets and a plurality of water outlets, the material of the water outlet being a pro
  • the water material is adsorbed as a circular or elliptical shape, and the water outlet has an inner diameter of 0.1 mm to 2 mm and an outer diameter of 0.5 mm to 5 mm to accommodate the width of the strip-shaped hydrophilic region.
  • the liquid adding device comprises a plurality of liquid adding pipes and a liquid adding pump
  • the liquid adding pump has a plurality of water outlets corresponding to the plurality of liquid feeding pipes
  • each of the water feeding ports of the liquid adding pump has a plurality of water outlets
  • the water inlets of the liquid adding pipes are respectively connected to each other to form a plurality of liquid adding channels
  • the water inlet of the liquid adding pump serves as a water inlet of the liquid adding device
  • the water outlet of the liquid adding pipe serves as the liquid adding device The outlet.
  • the dosing pump is a syringe pump or a peristaltic pump.
  • the peristaltic pump is a flow type peristaltic pump.
  • the liquid adding device further includes a fixing rod having a plurality of fixing portions for fixing the plurality of liquid filling tubes on the same straight line.
  • the fixing portion is a fixing hole that cooperates with the liquid filling tube, and the fixing hole is sleeved on an outer circumference of the liquid filling tube.
  • the liquid addition tube is a hydrophilic material.
  • the water outlet of the liquid adding device has a hydrophilic coating
  • the hydrophilic coating is polyvinylpyrrolidone.
  • the surface of the reaction device has a plurality of parallel strip-shaped hydrophilic regions spaced by a hydrophobic region, and the adsorption force of the hydrophilic region and the surface tension of the water itself are combined with the target antibody in the solution to be tested, and the separation by the hydrophobic region is prevented.
  • the reaction solution leaks out, and only the reaction liquid below the milliliter level can be used to complete the immunoblot detection, thereby saving more reagents and being suitable for trace detection;
  • Different target antibodies in the plurality of test solutions can be bound to the binding regions of different strips of hydrophilic regions of the same reaction device, so that the reaction is more suitable under the same conditions, and it is easy to carry out parallel control, which is improved.
  • the accuracy of the detection at the same time, the structure of the automatic detection system is simplified, and the production cost is reduced;
  • the mechanical motion device can be used to move the reaction device or the liquid addition device, so that the water outlet of the liquid addition channel moves relative to the strip-shaped hydrophilic region to promote the reaction efficiency of the reaction solution and the binding zone, thereby saving the immunoblot detection. time;
  • the preliminary drying can be achieved by simple suction of the waste liquid collecting device, or the surface of the immunoblotting reel can be quickly dried by simply integrating a simple drying device, and it is easier to carry out the in situ Detection, and further saving reaction time;
  • the automatic detection system also integrates a signal analysis device, which can obtain the detection signal of the immunoblot in situ without moving the reaction device, which simplifies the operation;
  • the alarm device can directly obtain the reaction end alarm or the reaction failure alarm of the immunoblot by the detection signal of the immunoblot, thereby avoiding the error of human observation, and at the same time, since the alarm device can automatically prompt the reaction to end, avoiding excessive reaction and causing The test results are not accurate;
  • the ultrasonic probe further accelerates the reaction speed of the immunoblotting, so that the whole reaction process of the immunoblotting is reduced by 30% to 50% compared with the conventional method, and the detection efficiency is greatly improved.
  • the liquid adding device can not only add the reaction liquid to the immunoblotting film, but also the hydrophilic water outlet can adsorb the reaction liquid, and use the surface tension of the water to reciprocate the reaction liquid along the strip-shaped hydrophilic region, thereby improving the reaction efficiency and saving.
  • the adsorption of the reaction liquid by the hydrophilic water outlet of the liquid adding device prevents the leakage of the reaction liquid, further ensuring that only the reaction liquid of the milliliter level or less is required in the immunoblot detection process, thereby saving the reagent and being suitable for trace detection. ;
  • the fixing rod can ensure that a plurality of liquid filling tubes are fixed on the same straight line, so that the immune reactions of the plurality of strip-shaped hydrophilic regions can be simultaneously performed, thereby ensuring parallel detection, thereby reducing operational errors.
  • the water outlet of the liquid adding device has a hydrophilic coating, so that the manufacturing material of the liquid adding device is not limited to a hydrophilic material. Instead, a relatively inexpensive hydrophobic material such as polypropylene can be used to facilitate the use of the liquid addition device or its sub-components as a disposable device, thereby avoiding the trouble of cleaning the instrument.
  • a relatively inexpensive hydrophobic material such as polypropylene can be used to facilitate the use of the liquid addition device or its sub-components as a disposable device, thereby avoiding the trouble of cleaning the instrument.
  • Figure 1 is a plan view of Embodiment 1 of the present invention.
  • Embodiment 1 of the present invention is a schematic diagram of a manufacturing process of Embodiment 1 of the present invention.
  • Embodiment 2 of the present invention is a schematic diagram of a manufacturing process of Embodiment 2 of the present invention.
  • Embodiment 4 is a schematic diagram of a manufacturing process of Embodiment 3 of the present invention.
  • Figure 5 is a schematic structural view of Embodiment 4 of the present invention.
  • Figure 6 is a schematic structural view of Embodiment 5 of the present invention.
  • Figure 7a is a front elevational view of a liquid adding device according to Embodiment 6 of the present invention.
  • Figure 7b is a side view of Embodiment 6 of the present invention.
  • Figure 7c is a front elevational view of a signal analysis apparatus according to Embodiment 6 of the present invention.
  • the invention discloses an automatic detection system for immunoblotting, comprising a reaction device, a liquid adding device 2 and a mechanical moving device;
  • the surface of the reaction device has a plurality of parallel strip-shaped hydrophilic regions 122 spaced by a hydrophobic region 121, each strip-shaped hydrophilic region 122 having one or more binding regions 122a along its length, the dosing
  • the device 2 has a plurality of liquid addition channels corresponding to the strip-shaped hydrophilic region 122, the control end of the mechanical motion device is connected to the reaction device or the liquid addition device 2;
  • Each of the strip-shaped hydrophilic regions 122 is used to bind different solutions to be tested, thereby independently performing immunoblot detection of the target antibody in different test solutions, and the binding region 122a has the target antigen for Binding to a target antibody in the solution to be tested;
  • the dosing device 2 is for adding a reaction solution to the strip-shaped hydrophilic region 122,
  • the mechanical motion device is configured to control the relative movement of the water outlet of the liquid addition channel along the strip-shaped hydrophilic region 122 to promote the relative movement of the reaction region 122a of the reaction liquid and the strip-shaped hydrophilic region 122 at a speed of 15 cm/s or less, thereby indirectly It plays a role of promoting the reaction reaction between the reaction liquid and the strip-shaped hydrophilic region 122.
  • the reaction liquid can still be slowly added to the surface of the strip-shaped hydrophilic region 122 through the liquid addition device 2.
  • the reaction solution includes a solution to be tested, a buffer solution, a secondary antibody solution or a color developing solution.
  • the reaction device comprises a substrate 11 having a plurality of parallel strip-shaped hydrophilic regions 122 separated by a hydrophobic region 121, and an immunoblotting film 12, which is subjected to vacuum adsorption, pasting, magnetic adsorption. Covered and fixed to the surface of the substrate 11 by means of a snap or the like.
  • the substrate 11 may be a flat substrate 11 or a circular substrate 11, which determines the morphology of the corresponding reaction device.
  • the immunoblotting film 12 is disposed above the flat substrate 11, and the liquid adding device 2 is disposed above the surface of the immunoblotting film 12, and the strips are disposed on both sides of the reaction device.
  • the linear motor 6 parallel to the water region 122 serves as a mechanical movement device and has a slide rail matched with the linear motor 6; the control end of the linear motor 6 is connected to the liquid adding device 2 for controlling the liquid addition passage along the strip-shaped hydrophilic region 122 The direction of reciprocation.
  • the width is 2 cm to 6 cm so as to be compatible with the reaction region of the immunoblotting film 12, and the immunoblotting film 12 is provided on the circumferential surface of the circular-axis substrate 11, immunizing
  • the strip-shaped hydrophilic region 122 of the blotting film 12 is perpendicular to the central axis of the cylindrical substrate 11, and the liquid adding device 2 is disposed opposite to the strip-shaped hydrophilic region 122 of the immunoblotting film 12; the cylindrical substrate 11 and
  • the rotary electric machine as a mechanical motion device is connected to rotate around its central axis under the control of the rotary electric machine, so that the strip-shaped hydrophilic region 122 of the immunoblotting film 12 also rotates around the central axis, thereby causing the liquid addition channel to follow the strip shape
  • the direction of the hydrophilic region 122 is relatively moved; considering the volume of the automatic detecting system and the flexibility of the motion control of the rotating electrical machine, the diameter of the cylindrical substrate 11 is preferably 2 cm to 6 cm.
  • the distance between the water outlet of the liquid addition channel and the corresponding strip hydrophilic region 122 is usually 0.1 mm to 2 mm to ensure that the liquid addition device 2 is added under the action of the surface tension of the water during the liquid addition or immunoblotting reaction.
  • a reaction liquid is adsorbed between the water outlet of the liquid passage and the corresponding strip-shaped hydrophilic region 122 (when the liquid addition passage is disposed above the strip-shaped hydrophilic region 122, the distance may be further than 2 mm).
  • the liquid adding device 2 can use a separate disposable liquid feeding tube 21, the water inlet of the liquid adding tube 21 is disposed outside, and the water outlet is disposed opposite to the strip-shaped hydrophilic region 122, and the water inlet can be opposite to the water outlet.
  • the reaction solution is added to the hydrophilic region 122; the liquid addition device 2 may also be composed of a liquid addition tube 21 and a liquid addition pump, and the liquid addition tube 21 and the liquid addition pump are connected to each other to form a one-to-one correspondence with the strip-shaped hydrophilic regions 122.
  • the water inlet of the dosing pump is used as The water inlet of the liquid adding device 2
  • the water outlet of the liquid adding pipe 21 serves as the water outlet of the liquid adding device 2
  • the liquid adding pump can use a syringe pump or a peristaltic pump, and the flow type peristaltic pump can
  • the reaction liquid is input to the plurality of liquid filling tubes 21 at a time, and the flow rate is at least 1 ⁇ l/min, which saves the solution to be tested.
  • the flow rate can reach 1000 ml/min, thereby maintaining a high level. The cleaning efficiency is thus particularly suitable for use in the present invention.
  • a waste liquid collection device 5 may also be provided to further accelerate the reaction efficiency of the immunoblotting.
  • a drying device may also be provided to further accelerate the reaction efficiency of the immunoblotting.
  • the waste liquid collection device 5 may include a liquid collection tube and a vacuum pump; the water inlet of the liquid collection tube serves as a water inlet of the waste liquid collection device 5, and the water outlet is connected to a vacuum pump, and the liquid collection tube is used to collect excess reaction.
  • the vacuum pump is used to pump the reaction liquid.
  • the waste liquid collection device 5 can also include a drainage tube.
  • a liquid collection tank 52 the water inlet of the drainage tube serves as a water inlet of the waste liquid collection device 5, and the water outlet is disposed below the water inlet, and the liquid collection tank 52 is disposed at the water outlet of the drainage tube.
  • the drainage tube is used to extract excess reaction liquid
  • the liquid collection tank 52 is used to collect excess reaction liquid; in this way, the reaction liquid is difficult to be completely introduced into the liquid collection tank 52, and generally needs to be additionally set.
  • an auxiliary drying device drying the reaction apparatus; drying apparatus, a fan or blower, disposed opposite the reaction surface of the device, the wind speed is set to 2m 3 / min ⁇ 5m 3 / min, the reaction apparatus in a uniform drying while avoiding Means an antigen or antibody to be modified surface; a plurality of ultrasonic probe 3, 122 correspond to emit ultrasonic signals similar to the strip 122 and strip-shaped hydrophilic areas hydrophilic areas, means to accelerate the reaction rate of the surface reaction.
  • the signal analysis device 4 can also be directly provided for acquiring the detection signal of the immunoblot; generally, the signal analysis device 4 includes the light source 42 and the image sensor 41, and the image sensor 41 It may be linear, which is perpendicular to the strip-shaped hydrophilic region 122; according to the marking of the secondary antibody in the secondary antibody solution, the light source 42 may be selected from a laser light source 42 or a visible light source 42 (for example, the maximum absorption of the commonly labeled fluorescein isothiocyanate) The wavelength of light is 490 nm to 495 nm, and the maximum absorption wavelength of tetraethylrhodamine is 570 nm), which is used to emit visible light or laser light toward the reaction device, and forms an immunoblot detection signal on the surface of the reaction device, whether it is visible light or laser light.
  • the shape of the light source 42 is preferably elongated, which is also perpendicular to the direction of the strip-shaped hydrophilic region 122, so as to ensure uniformity of light projected to the same position of each strip-shaped hydrophilic region 122, so as to avoid contrast errors;
  • the image sensor 41 is used to acquire a detection signal of an immunoblot.
  • the substrate 11 is a circular-arc substrate 11, the outer strip is hydrophilic.
  • the field 122 is actually circular, and the image sensor 41 needs to take multiple shots to obtain a complete detection signal. Since the strip-shaped hydrophilic region 122 has a detection signal only in the joint region 122a, the trigger condition of the image sensor 41 can be set.
  • the image of the detection signal acquired by the image sensor 41 can be directly input into the data processor, and the detection signal of some standard samples is stored in the data processor, The detection signal of the solution to be tested is compared with the detection signal of the standard sample, and the detection results of the type and concentration of the target antibody in the solution to be tested can be obtained.
  • the liquid addition device 2 and the ultrasonic probe 3 or the signal analysis device 4 and the like may be fixed on a bracket and disposed on the surface of the reaction device.
  • a folding rod or a telescopic rod may be provided to the liquid adding device 2 and each additional device to adjust the relative position and distance of the liquidizing device and the reaction device, respectively.
  • the binding region 122a can be divided into an experimental region and a control region, and the experimental region is used for binding to a target protein, and the control region can be classified into a positive control region according to its function.
  • a negative control zone or a conjugate control zone wherein the positive control zone usually contains an antigen that can bind to other components than the target antibody in the solution to be tested, for example, when blood is used as the solution to be tested, the target antibody of the positive control zone In principle, serum antibodies can be used.
  • the color of the positive control area indicates that the surface of the reaction device has not deteriorated, and the type of the solution to be tested is correct.
  • the negative control area can be coated with unlabeled secondary antibody, so that the negative control area cannot be colored under standard conditions. If it develops color, it may prove that there may be abnormalities such as surface deterioration of the reaction device, and the immunoblotting film 12 should be replaced and re-measured; and the target antigen of the conjugate control region is an IgG conjugate, an IgM conjugate or an IgA conjugate due to IgG. , IgM and IgA are common immunoglobulins, and the presence of a conjugate control region can indicate whether the antibody of interest has been successfully bound.
  • a control solution may be added to the liquid addition channel corresponding to one of the strip-shaped hydrophilic regions 122 for indicating the end of the reaction of the immunoblotting or the reaction failure; at this time, an alarm device may be additionally provided, and the input end thereof is connected to the signal analysis device 4 At the output end, the reaction end detection alarm or the reaction failure alarm is directly obtained through the detection result of the control region or the strip-shaped hydrophilic region 122 added to the control solution, thereby avoiding the error of human observation, and at the same time, the alarm device can automatically prompt The reaction is over and the detection results caused by overreaction are avoided.
  • the preparation method of the immunoblot film 12 used in the above automatic detection system can be roughly classified into two types:
  • the first type binding the target antigen on the hydrophobic membrane to form the same binding band as the target antigen, the material of the hydrophobic membrane is nitrocellulose, nylon or polyvinylidene fluoride; forming a hydrophobic film on the hydrophobic membrane
  • the hydrophobic region 121 is configured to separate the hydrophobic membrane into a plurality of strip-shaped hydrophilic regions 122 while the binding zone corresponds to the binding zone 122a of the binding zone such that each of the strip-shaped hydrophilic regions 122 has the same number of target antigens Bonding zone 122a; the hydrophobic coating needs to be non-toxic and can be dried below 37 degrees to avoid affecting the activity of the target antigen, such as the hydrophobic coating in Chinese patent document CN201380057250.
  • a plurality of strip-shaped hydrophilic films are attached in parallel to the hydrophobic film such that the hydrophobic film has a strip-shaped hydrophilic region 122 formed with a plurality of strip-shaped hydrophilic films, the strip-shaped hydrophilic The regions 122 are spaced apart by the hydrophobic region 121; the surface of the hydrophobic film may be provided with a groove matching the strip-shaped hydrophilic film to be thin with the strips
  • the film is bonded and fixed such that the surface of the strip-shaped hydrophilic region 122 is lower than the surface on which the hydrophobic layer is located, which further ensures that the reaction liquid between the strip-shaped hydrophilic regions 122 does not interfere with each other.
  • the surface of the immunoblotting film 12 obtained by the above method has a plurality of strip-shaped hydrophilic regions 122 spaced apart by the hydrophobic region 121, and the width of the hydrophobic region 121 and the strip-shaped hydrophilic region 122 is about 1 mm to 5 mm, while saving the reaction liquid.
  • the detection signal can be obtained sufficiently accurately, and the material of the strip-shaped hydrophilic region 122 is nitrocellulose, nylon or polyvinylidene fluoride to bind the target antigen, and the hydrophobic region 121 is usually an organic hydrophobic polymer having a silane group. It is also possible to carry a siloxy group or a silanol group such as polydimethylsiloxane or a derivative thereof.
  • the material of the water outlet of the liquid adding device 2 used in the above automatic detecting system is a hydrophilic material for adsorbing the reaction liquid, and the shape is circular or elliptical, and the inner diameter of the water outlet is 0.1 mm to 2 mm, and the outer diameter is 0.5.
  • the reaction liquid can completely cover the strip
  • the liquid adding device 2 can use a single disposable filling pipe 21, the water inlet of the liquid adding pipe 21 is located above, and the water outlet is located below, from the upper water inlet, to the strip-shaped hydrophilic region 122 opposite to the water outlet.
  • the reaction liquid is added; the liquid adding device 2 can also be composed of a plurality of liquid adding tubes 21 and a liquid adding pump, and when different types of reaction liquids are added, the liquid filling tubes 21 of different inner diameters can be used to adapt to the experimental requirements, for example, When the buffer is added, the larger inner diameter filling tube 21 can be replaced to clean the strip-shaped hydrophilic region 122.
  • the liquid-filling device 2 can also use a fixing rod 23 having a plurality of fixing portions to fix the plurality of liquid-filling tubes 21 on the same straight line; the fixing portion can be a fixing hole that is sleeved on the outer circumference of the liquid-filling tube 21, A fixing structure such as a clip or a buckle is shown in FIG. 2; at this time, the mechanical moving device can move the liquid adding device 2 as a whole by moving the fixing rod 23.
  • the liquid adding device 2 can use a relatively inexpensive material such as polypropylene, and the sub-components of the liquid adding device 2 or the liquid adding device 2 can be conveniently used as a disposable device, thereby avoiding the trouble of cleaning the instrument; when the liquid adding device 2 is hydrophobic once
  • the water outlet can be coated with a hydrophilic coating such as polyvinylpyrrolidone, poly N-vinyl butyrolactam, poly N-vinylcaprolactam, polyvinyl alcohol, polyvinyl alcohol derivative, polyurethane or modified Polyethylene, etc.
  • Example 1 is an immunoblot film 12 according to Example 1 of the present invention, which is made of a nylon membrane; the surface of the immunoblotting film 12 has a plurality of strip-shaped hydrophilic regions 122 spaced apart by a hydrophobic region 121, and the strip-shaped hydrophilic regions 122 And the width of the hydrophobic region 121 is 1 mm, and the strip-shaped hydrophilic region 122 has one or more bonding regions 122a.
  • the binding region 122a has a target antigen;
  • the preparation method of the immunoblot film 12 is as shown in FIG. 2, firstly, four target antigens are bound on a hydrophobic membrane of nylon to form four binding bands, as shown in FIG. 2a-b; then a hydrophobic coating is formed on the hydrophobic membrane.
  • the hydrophobic region 121 is configured to separate the hydrophobic membrane into a plurality of strip-shaped hydrophilic regions 122 while separating the binding bands into the same number of binding regions 122a as the binding bands, such that each of the strip-shaped hydrophilic regions 122 has a target antigen
  • the same number of binding regions 122a as shown in Fig. 2c, obtain the immunoblotting film 12 shown in Fig. 1.
  • composition of the hydrophobic coating is 1.448 wt% of tetraethyl orthosilicate, 0.1588 wt% of dodecyltrimethoxysilane, 0.0698 wt% of ammonium hydroxide, 0.1938 wt% of HCl, 208 wt% of ethanol and 78.1198 wt. % of softened water.
  • FIG. 3 is a schematic view showing a process of fabricating the immunoblotting film 12 according to Example 2 of the present invention, in which a substrate 11 of polydimethylsiloxane (PDMS) is first placed, and then a nitrocellulose having a plurality of bonding regions 122a and having a width of 3 mm is used. The plain films were adhered to the substrate 11 at intervals of 3 mm.
  • PDMS polydimethylsiloxane
  • Embodiment 4 is a schematic view showing a process of fabricating the immunoblotting film 12 according to Embodiment 3 of the present invention, which differs from Embodiment 2 in that a 5 mm wide groove and a width of 5 mm are formed on the PDMS substrate 11 at intervals of 5 mm.
  • the vinylidene fluoride film can be just stuck in the groove.
  • Fig. 5 is a schematic view showing the structure of the immunoblotting film 12 of Example 4 of the present invention, which differs from Example 1 in that an immunoblotting film 12 having a length of 10 cm is wound on a circular-arc substrate 11 having a diameter of about 3.2 cm.
  • Embodiment 5 is an automatic detection system, including a reaction device, a liquid addition device 2, an ultrasonic probe 3, a signal analysis device 4, a waste liquid collection device 5, and the like; wherein the reaction device is in Embodiment 4
  • the relative movement along the strip-shaped hydrophilic region 122 since the distance between the water outlet of the liquid addition channel along the strip-shaped hydrophilic region 122 is only 0.1 mm to 2 mm, the reaction liquid is actually adsorbed between the two, and the relative of the two
  • the movement indirectly acts to promote the mixed reaction of the reaction liquid and the strip-shaped hydrophilic region 122; at the same time, the ultrasonic probe 3 disposed opposite to the strip-shaped hydrophilic region 122 can be used to emit an ultrasonic
  • the above automatic detection system can be applied to the immunoblot detection, and the indirect method is used for the chemiluminescence detection as an example, and specifically includes the following steps:
  • the plurality of test solutions are passed through the liquid adding device 2 to be added to the surface of the different strip-shaped hydrophilic regions 122, so that the target antibody in the solution to be tested is completely combined with the target antigen on the binding band, and at the same time, the ultrasonic probe 3 emits
  • the ultrasonic signal accelerates the reaction speed of the surface of the reaction device; the rotating motor is in operation at this time, so that the reaction device rotates at a linear velocity of 15 cm/s or less for 30 min to 60 min; while the solution to be tested can still be slowly added through the dosing device 2
  • the surface of the strip-shaped hydrophilic region 122 is prevented from evaporating to maintain the liquid level on the surface of the strip-shaped hydrophilic region 122 from 0.1 mm to 1 mm; in this case, even if the liquid filling device 2 remains
  • the solution to be tested is counted, and the required volume of the reaction solution is only required to be tens of microliters to the milliliter level;
  • the vacuum pump suctions the excess solution to be tested on the surface of the strip-shaped hydrophilic region 122 through the collecting tube, and then the surface of the strip-shaped hydrophilic region 122 is dried by vacuum pumping, or assisted by an additional drying device. Dry it;
  • the solution to be tested in the original dosing device 2 is replaced by a buffer, and the solution to be tested on the surface of the immunobloted film 12 is washed for about 5 min to 10 min; since the buffer is usually PBS or the like.
  • the lower cost solution may be set to a flow rate exceeding 10 ml/min or more with respect to the solution to be tested, and more volume is added;
  • the immunoblot detection result is obtained by the signal analysis device 4.
  • the embodiment provides an automatic detection system, including a flat substrate 11, an immunoblot film 12, a liquid adding device 2, a signal analyzing device 4, a waste liquid collecting device 5, and the like;
  • the immunoblotting film 12 is disposed above the flat substrate 11, and the surface of the immunoblotting film 12 has a plurality of parallel strip-shaped hydrophilic regions 122 spaced apart by a hydrophobic region 121, and each of the strip-shaped hydrophilic regions 122 Used to combine different test solutions, respectively, to independently perform immunoblotting of target antibodies in different solutions to be tested Measurement;
  • the liquid adding device 2 is composed of a liquid adding pipe 21, a fixing rod 23, and a flow type peristaltic liquid pump, and the liquid adding pump has a plurality of water outlets corresponding to the plurality of liquid adding pipes 21, each of the liquid adding pumps
  • the water inlet communicates with the water inlets of the plurality of liquid filling tubes 21 to form a plurality of liquid adding passages corresponding to the strip-shaped hydrophilic regions 122;
  • the fixing rod 23 has a plurality of fixing holes, and is sleeved on the outer circumference of the liquid filling tube 21.
  • a linear motor 6 is disposed on both sides of the liquid adding device 2, and a control end of the linear motor 6 is connected to the fixing rod 23 of the liquid adding device 2, and the linear motors 6 on both sides are synchronized; while the linear motor 6 moves the liquid adding device 2,
  • the solution to be tested can still be slowly added to the surface of the strip-shaped hydrophilic region 122 by the dosing device 2 to prevent evaporation of the liquid to maintain the liquid level on the strip-shaped hydrophilic region 122 from 0.1 mm to 1 mm.
  • the waste liquid collection device 5 includes an inverted L-shaped drainage tube 51 and a sump 52, as shown in FIG. 7b; the water inlet of the inverted L-shaped drainage tube 51 serves as a water inlet of the waste liquid collection device 5 The water inlet is disposed below the water inlet, the liquid collection tank 52 is disposed below the water outlet of the inverted L-shaped drainage tube 51, and the inverted L-shaped drainage tube 51 is used to extract excess reaction liquid, the collection The liquid pool 52 is used to collect excess reaction liquid.
  • a signal analyzing device 4 is disposed above the immunoblotting film 12 for acquiring a detection signal of the immunoblotting, and since the immunoblotting film 12 is developed into a planar structure in the present embodiment, as shown in FIG. 7c, the signal analyzing device 4 Once the signal is acquired, all the detection signals can be obtained.
  • the detection principle of the automatic detection system for the immunoblotting is similar to that of the embodiment 5, except that in the fifth embodiment, the rotary electric machine controls the reaction device to perform a rotational motion, and in the present embodiment, the reciprocating motor controls the liquid addition device 2 to reciprocate.
  • the movement also serves to promote the reaction of the reaction liquid flowing out of the liquid addition device 2 with the surface of the strip-shaped hydrophilic region 122.

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Engineering & Computer Science (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

La présente invention concerne un système de détection automatique qui comprend un dispositif de réaction, un dispositif d'ajout de liquide (2), et un dispositif de mouvement mécanique. Des régions hydrophiles multiples en forme de bandes parallèles (122) séparées par des régions hydrophobes (121) sont agencées sur la surface du dispositif de réaction. Chaque région hydrophile en forme de bande (122) comporte une ou plusieurs régions de conjonction (122a) dans la direction de la longueur de la région hydrophile en forme de bande (122). Le dispositif d'ajout de liquide (2) est pourvu de canaux d'ajout de liquide multiples dans une correspondance un à un avec les régions hydrophiles en forme de bande (122). Une extrémité de commande du dispositif de mouvement mécanique est raccordée au dispositif de réaction ou au dispositif d'ajout de liquide (2). Le dispositif d'ajout de liquide (2) est utilisé pour ajouter un liquide de réaction aux régions hydrophiles en forme de bande (122). Le dispositif de mouvement mécanique est utilisé pour commander une sortie d'eau du canal d'ajout de liquide de façon à se déplacer par rapport aux régions hydrophiles en forme de bande (122), de façon à favoriser le mouvement relatif entre le liquide de réaction et les régions de conjonction (122a) des régions hydrophiles en forme de bande (122). La structure du système de détection automatique est simplifiée, une détection in situ peut être mise en œuvre, le système de détection automatique peut être lié à un anticorps cible au moyen de la force d'adsorption des régions hydrophiles en forme de bande (122) et, par conséquent, la consommation d'un réactif est réduite, et le système de détection automatique est adapté pour la détection de trace.
PCT/CN2017/078576 2017-03-27 2017-03-29 Système de détection automatique pour immunotransfert WO2018176262A1 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
CN201710190190.1A CN107045067B (zh) 2017-03-27 2017-03-27 一种用于免疫印迹的加液装置
CN201710189295.5A CN106932596A (zh) 2017-03-27 2017-03-27 一种免疫印迹薄膜及其制备方法
CN201710189318.2 2017-03-27
CN201710190186.5A CN107045066A (zh) 2017-03-27 2017-03-27 一种免疫印迹卷轴
CN201710190190.1 2017-03-27
CN201710189295.5 2017-03-27
CN201710189318.2A CN106680514B (zh) 2017-03-27 2017-03-27 一种用于免疫印迹的自动检测器
CN201710190186.5 2017-03-27
CN201710188888.X 2017-03-27
CN201710188888.XA CN106814199B (zh) 2017-03-27 2017-03-27 一种免疫印迹的自动检测系统

Publications (1)

Publication Number Publication Date
WO2018176262A1 true WO2018176262A1 (fr) 2018-10-04

Family

ID=63674013

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/078576 WO2018176262A1 (fr) 2017-03-27 2017-03-29 Système de détection automatique pour immunotransfert

Country Status (1)

Country Link
WO (1) WO2018176262A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022172289A1 (fr) * 2021-02-10 2022-08-18 Anupama Chaudhary Système et procédé pour automatiser un sondage de chimioluminescence manuel en immunotransfert

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031979A1 (fr) * 2001-10-05 2003-04-17 Surmodics, Inc. Jeux d'echantillons ordonnes de façon aleatoire et procedes de fabrication et d'utilisation
CN102445375A (zh) * 2010-09-30 2012-05-09 广州阳普医疗科技股份有限公司 加样皿装置
CN102740978A (zh) * 2009-12-18 2012-10-17 扎芬纳股份公司 微量移液管
CN103261872A (zh) * 2010-10-06 2013-08-21 保科医疗公司 生物样品的有效处理方法和系统
CN103954785A (zh) * 2014-03-20 2014-07-30 欧蒙医学诊断（中国）有限公司 一种蛋白印迹分析仪的液体系统
CN104769429A (zh) * 2012-06-11 2015-07-08 Abo血型诊断公司 体外诊断装置及其使用
CN105527282A (zh) * 2014-10-16 2016-04-27 郑兆珉 一种布基生化检测装置及其制作方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031979A1 (fr) * 2001-10-05 2003-04-17 Surmodics, Inc. Jeux d'echantillons ordonnes de façon aleatoire et procedes de fabrication et d'utilisation
CN102740978A (zh) * 2009-12-18 2012-10-17 扎芬纳股份公司 微量移液管
CN102445375A (zh) * 2010-09-30 2012-05-09 广州阳普医疗科技股份有限公司 加样皿装置
CN103261872A (zh) * 2010-10-06 2013-08-21 保科医疗公司 生物样品的有效处理方法和系统
CN104769429A (zh) * 2012-06-11 2015-07-08 Abo血型诊断公司 体外诊断装置及其使用
CN103954785A (zh) * 2014-03-20 2014-07-30 欧蒙医学诊断（中国）有限公司 一种蛋白印迹分析仪的液体系统
CN105527282A (zh) * 2014-10-16 2016-04-27 郑兆珉 一种布基生化检测装置及其制作方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022172289A1 (fr) * 2021-02-10 2022-08-18 Anupama Chaudhary Système et procédé pour automatiser un sondage de chimioluminescence manuel en immunotransfert

Similar Documents

Publication Publication Date Title
AU724089B2 (en) Sensor laminates and multi-sectioned fluid delivery devices for detecting by immunoassay target molecules in biological fluids
US7396674B2 (en) Reaction vessel
CN109142707B (zh) 一种荧光免疫层析测量装置
JPH01229969A (ja) 改良されたメンブレン支持形式の免疫分析法
CN102680545B (zh) 用于检测电解质项目和总二氧化碳的测试仪器
CN111656198B (zh) 样本测定装置、试剂容器以及样本测定方法
US10191037B2 (en) Methods of and systems for improved detection sensitivity of assays
JP2013519899A (ja) 抗体及び抗原を検出するためのイムノアッセイデバイス
JPH03170060A (ja) 生体液中の分子状検体の試験装置および血液中の所定分子の試験方法
WO2017156910A1 (fr) Procédé et dispositif de détection de centrifugation
WO2018176262A1 (fr) Système de détection automatique pour immunotransfert
CN113533736A (zh) 肺炎支原体IgM抗体胶体金免疫层析检测试剂盒及其制备方法
WO2016031899A1 (fr) Capuchon de buse pour récipient d'essai d'échantillon, et récipient d' essai d'échantillon et kit d'essai d'échantillon l'utilisant
JP2013543981A5 (fr)
JP2013543981A (ja) ウェル内検量機能を印刷するための方法及びシステム
JP6184292B2 (ja) コンパクトディスク型マイクロチップを用いた分析装置
CN110007075A (zh) 一种检测试剂条及其应用
CN1595160A (zh) 硅烷交联壳聚糖膜基的流动注射化学发光免疫检测池及制备方法
JP6190472B2 (ja) 新規のＰｏＣ検査システムおよび方法
CN109100505A (zh) 一种定量检测血管内皮标志物cd105的免疫荧光试剂盒
JP4769171B2 (ja) 体液試料濾過分離装置及び方法
CN211179858U (zh) 一种血清淀粉样蛋白a层析检测卡
CN115112640A (zh) 一种检测系统和检测方法
CN206696297U (zh) 一种用于免疫印迹的加液装置
CN107045066A (zh) 一种免疫印迹卷轴

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17903889

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17903889

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 10/08/2020)

122 Ep: pct application non-entry in european phase

Ref document number: 17903889

Country of ref document: EP

Kind code of ref document: A1