WO2018176262A1

WO2018176262A1 - Automatic detection system for immunoblotting

Info

Publication number: WO2018176262A1
Application number: PCT/CN2017/078576
Authority: WO
Inventors: 黄世海
Original assignee: 武汉优视科技有限公司
Priority date: 2017-03-27
Filing date: 2017-03-29
Publication date: 2018-10-04

Abstract

An automatic detection system comprises a reaction device, a liquid adding device (2), and a mechanical motion device. Multiple parallel strip-shaped hydrophilic regions (122) separated by hydrophobic regions (121) are arranged on the surface of the reaction device. Each strip-shaped hydrophilic region (122) has one or more conjunction regions (122a) in the length direction of the strip-shaped hydrophilic region (122). The liquid adding device (2) is provided with multiple liquid adding channels in a one-to-one correspondence with the strip-shaped hydrophilic regions (122). A control end of the mechanical motion device is connected to the reaction device or the liquid adding device (2). The liquid adding device (2) is used for adding a reaction liquid to the strip-shaped hydrophilic regions (122). The mechanical motion device is used for controlling a water outlet of the liquid adding channel to move relative to the strip-shaped hydrophilic regions (122), so as to promote the relative motion between the reaction liquid and the conjunction regions (122a) of the strip-shaped hydrophilic regions (122). The structure of the automatic detection system is simplified, in-situ detection can be implemented, the automatic detection system can be bound to a target antibody by means of the adsorption force of the strip-shaped hydrophilic regions (122), and accordingly, the consumption of a reagent is decreased, and the automatic detection system is suitable for trace detection.

Description

An immunoblot automatic detection system

Technical field

The invention belongs to the field of protein analysis, and more particularly to an automatic detection system for immunoblotting.

Background technique

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.

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. Therefore, it has the following disadvantages: First, due to the integration of the shaker for oscillating the reagent bottle, the structure of the entire analyzer is more complicated, thereby increasing the manufacturing cost; Second, the reaction conditions of different reagent bottles are slightly different, thereby affecting the immunoblotting Accuracy; Third, the film needs to be removed from the reagent bottle and dried before testing, so that in-situ detection cannot be achieved.

Summary of the invention

In view of the above drawbacks or improvements in the prior art, 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.

In order to achieve the above object, according to an aspect of the present invention, an automatic detection system for an immunoblot is provided, 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.

Preferably, 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.

Further preferably, the immunoblotting film is attached to the surface of the substrate.

Further preferably, 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.

Further preferably, the upper surface of the substrate is provided with a buckle that fixes the immunoblotting film to the upper surface of the substrate.

Further preferably, the substrate is in the form of a flat plate.

Further preferably, 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.

Still more preferably, both sides of the automatic detector have slide rails parallel to the strip-shaped hydrophilic regions.

Further preferably, the substrate has a circular axis shape, and the strip-shaped hydrophilic region is perpendicular to a central axis of the circular-axis substrate.

Still more preferably, the cylindrical substrate has a diameter of 2 cm to 6 cm and a width of 2 cm to 6 cm.

Still more preferably, 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.

Preferably, 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.

Preferably, 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, and the water outlet of the liquid adding pipe serves as a water outlet of the liquid adding device.

Preferably, the automatic detection system further includes a waste liquid collection device for collecting excess reaction liquid.

Further preferably, 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.

Further preferably, 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.

Preferably, the automatic detection system further comprises a drying device.

Further preferably, the drying device is a fan or an exhaust fan having an air volume of 2 m ³ /min to 5 m ³ /min.

Preferably, the automatic detection system further comprises an ultrasound probe disposed opposite the strip-shaped hydrophilic region for accelerating the reaction speed of the immunoblotting.

Preferably, 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.

Further preferably, 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.

Still more preferably, 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.

Further preferably, 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.

Further preferably, 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.

Preferably, 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.

According to another aspect of the present invention, there is further provided 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.

Preferably, the strip-shaped hydrophilic region has a plurality of binding regions, the plurality of binding regions including an experimental region and a control region.

As a further preferred, the control zone comprises a positive control zone, a negative control zone or a conjugate control zone.

Preferably, the material of the strip-shaped hydrophilic region is nitrocellulose, nylon or polyvinylidene fluoride.

Preferably, the hydrophobic region has a silane group.

Further preferably, the material of the hydrophobic region is polydimethylsiloxane or a derivative thereof.

Preferably, the strip-shaped hydrophilic region and the hydrophobic region have a width of 1 mm to 5 mm.

Preferably, the immunoblotting film is wound into a reel structure in the direction of the strip-shaped hydrophilic region.

Further preferably, the reel structure has a diameter of 2 cm to 6 cm and a width of 2 cm to 6 cm.

As a further preferred, the reel structure is wound on a circular shaft-shaped substrate.

According to another aspect of the present invention, there is also provided a method of preparing the above immunoblot film, comprising:

Binding the target antigen on the hydrophobic membrane to form the same binding band as the target antigen, 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.

And coating a plurality of strip-shaped hydrophilic films on the hydrophobic film in parallel such that the hydrophobic film has a plurality of parallel strip-shaped hydrophilic regions formed with the plurality of strip-shaped hydrophilic films, the strip-shaped hydrophilic regions Spaced by hydrophobic regions.

Preferably, the surface of the hydrophobic film has a groove that cooperates with the strip-shaped hydrophilic film for fixing the strip-shaped hydrophilic film.

According to another aspect of the present invention, there is also provided 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.

Preferably, 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, and 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, and the water outlet of the liquid adding pipe serves as the liquid adding device The outlet.

Further preferably, the dosing pump is a syringe pump or a peristaltic pump.

As still further preferred, the peristaltic pump is a flow type peristaltic pump.

Further preferably, 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.

Further preferably, 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.

As a further preferred, the liquid addition tube is a hydrophilic material.

Preferably, the water outlet of the liquid adding device has a hydrophilic coating, and the hydrophilic coating is polyvinylpyrrolidone. Poly N-vinyl butyrolactam, poly N-vinylcaprolactam, polyvinyl alcohol, polyvinyl alcohol derivatives, polyurethane or modified polyethylene.

In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

1. 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;

2. 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;

3. 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;

4. Since only the reaction liquid below the milliliter level is needed, 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;

5. 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;

6. 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;

7. 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.

8. 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 time of immunoblot detection;

9. 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. ;

10. 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.

11. 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.

DRAWINGS

Figure 1 is a plan view of Embodiment 1 of the present invention;

2 is a schematic diagram of a manufacturing process of Embodiment 1 of the present invention;

3 is a schematic diagram of a manufacturing process of Embodiment 2 of the present invention;

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;

Throughout the drawings, the same reference numerals are used to refer to the same elements or structures, wherein: 11-substrate, 12-immunoblotted film, 121-hydrophobic region, 122-hydrophilic region, 122a-binding region, 2 - Dosing device, 21-plus pipe, 23-fixed rod, 3-ultrasonic probe, 4-signal analysis device, 41-image sensor, 42-light source, 5-waste collection device, 51-inverted L-type drainage tube , 52-pool, 6-linear motor.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Further, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

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. While the mechanical movement device moves the liquid addition device 2, the reaction liquid can still be slowly added to the surface of the strip-shaped hydrophilic region 122 through the liquid addition device 2. In order to prevent evaporation of the liquid to maintain the liquid level on the strip-shaped hydrophilic region 122 of 0.1 mm to 1 mm; the reaction solution includes a solution to be tested, a buffer solution, a secondary antibody solution or a color developing solution.

Wherein, 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. When the substrate 11 is a flat substrate 11, 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.

When the substrate 11 is a circular-arc substrate 11, 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. a plurality of liquid addition channels, 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, and 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 μl/min, which saves the solution to be tested. When the cleaning liquid such as buffer is introduced, 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.

In the automatic detection system, a waste liquid collection device 5, a drying device, and/or an ultrasonic probe 3 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. In this case, since the height of the reaction liquid is only 0.1 mm to 1 mm, the excess reaction liquid can be almost completely pumped away; the waste liquid collection device 5 can also include a drainage tube. And 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. Below, the drainage tube is used to extract excess reaction liquid, and 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.

Since the automatic detection system can realize in-situ reaction and drying, 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. 42 is required to have uniform 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. When 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. When it is opposite to the bonding area 122a, 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.

When the automatic detection system includes the ultrasonic probe 3 or the signal analysis device 4 and other fixed additional devices, 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. On the opposite side, on the bracket, 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.

When a strip-shaped hydrophilic region 122 has a plurality of binding regions 122a, 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.

Secondly, 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. Mm ~ 5mm, in order to adapt to the width of the strip-shaped hydrophilic region 122, since the water has a surface tension, even if the outer diameter of the water outlet is slightly different from the width of the strip-shaped hydrophilic region 122, the reaction liquid can completely cover the strip The width direction of the hydrophilic region 122; the material of the water outlet of the liquid adding device 2 is a hydrophilic material to adsorb the reaction liquid, and at the same time, the distance between the water outlet of the liquid adding device 2 and the corresponding strip-shaped hydrophilic region 122 It is 0.1 mm to 2 mm, and both of them are hydrophilic materials, so that leakage of the reaction liquid can be prevented to contaminate the adjacent strip-shaped hydrophilic regions 122.

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 For the material, 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

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. The 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.

Example 2

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.

Example 3

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.

Example 4

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.

Example 5

As shown in FIG. 6, 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 immunoblot film 12 wound on the circular-axis substrate 11; the control end of the rotating electrical machine is connected to the circular-arc substrate 11, and the reaction device is rotated at a linear velocity of 15 cm/s or less to make the water inlet of the liquid-filling channel 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 signal to accelerate the reaction efficiency; The waste liquid collection device 5 at the bottom can be used to collect excess reaction liquid; the waste liquid collection device 5 of the embodiment includes a liquid collection tube and a vacuum pump, one end of the liquid collection tube is 1220.1 mm to 2 mm from the strip-shaped hydrophilic region, and the other end is connected. vacuum .

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:

S1. 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;

S2. After the reaction is completed, 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;

S3. In the same manner as in S1-S2., 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;

S4. In the same manner as in S1-S2., the buffer solution in the original dosing device 2 is replaced by a secondary antibody solution, so that the secondary antibody completely binds to the target antibody;

S5. In the same manner as in S3., replace the secondary antibody solution in the original dosing device 2 with a buffer solution, and rinse the second antibody solution on the surface of the immunoblot film 12;

S6. In the same manner as in S1-S2., replacing the buffer in the original dosing device 2 with a color developing solution, so that the labeled secondary antibody emits light;

S7. In the same manner as in S3., replacing the color developing solution in the original dosing device 2 with a buffer solution, and rinsing the color developing solution on the surface of the immunoblotting film 12;

S8. The immunoblot detection result is obtained by the signal analysis device 4.

Example 6

As shown in FIG. 7, 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. To fix the plurality of liquid filling tubes 21 on the same straight line as shown in Fig. 7a.

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.

Those skilled in the art will appreciate that the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and scope of the present invention, All should be included in the scope of protection of the present invention.

Claims

An automatic detection system for immunoblotting, comprising: a reaction device, a liquid adding device (2) and a mechanical motion device;

The reaction device surface has a plurality of parallel strip-shaped hydrophilic regions (122) spaced apart by a hydrophobic region (121), each strip-shaped hydrophilic region (122) having one or more bonding regions along its length direction (122a), the liquid adding device (2) has a plurality of liquid addition channels corresponding to the strip-shaped hydrophilic regions (122), and the control end of the mechanical motion device is connected to the reaction device or the liquid adding 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 solutions to be tested, and the binding region (122a) has a target An antigen for binding to a target antibody in a solution to be tested; the dosing device (2) for adding a reaction solution to the strip hydrophilic region (122) for controlling the outflow of the liquid addition channel The nozzle moves relative to the strip-shaped hydrophilic region (122) to promote relative movement of the reaction zone (122a) of the strip-like hydrophilic region (122); the reaction solution includes a solution to be tested, a buffer solution, and a secondary antibody. Solution or color solution.
The automatic detection system according to claim 1, wherein said reaction device comprises a substrate (11) and an immunoblotting film (12), said immunoblotting film (12) covering said substrate (11) The surface, the immunoblot film (12) has a plurality of parallel strip-shaped hydrophilic regions (122) spaced apart by a hydrophobic region (121).
The automatic detection system according to claim 2, characterized in that the substrate (11) is in the form of a flat plate or a circular shaft.
The automatic detecting system according to claim 3, wherein said substrate (11) is in the form of a flat plate, and said mechanical moving means is a linear motor (6) disposed on both sides of the immunoblotting film (12). The control end of the linear motor (6) is connected to the liquid adding device (2) for controlling the reciprocating movement of the liquid adding passage in the direction of the strip-shaped hydrophilic region (122).
The automatic detecting system according to claim 3, wherein said substrate (11) has a circular axis shape, said mechanical moving device is a rotating electrical machine, and said control end of said rotating electrical machine is connected to said circular shaft shape The substrate (11) is controlled to rotate the immunoblot film (12) about the central axis of the substrate (11).
The automatic detection system according to claim 1, wherein said automatic detection system further comprises a waste liquid collection device (5) for collecting excess reaction liquid.
The automatic detection system according to claim 6, wherein the waste liquid collection device (5) comprises a plurality of liquid collection tubes and a vacuum pump; the water inlets of the plurality of liquid collection tubes are respectively associated with a plurality of strips The water area (122) is opposite to serve as the water inlet of the waste liquid collecting device (5), and the water outlet is connected to a vacuum pump for collecting strip-shaped hydrophilic The surface (122) has excess reaction liquid on the surface, and the vacuum pump is used to pump the reaction liquid.
The automatic detection system according to claim 6, wherein said waste liquid collection device (5) comprises a plurality of drainage tubes (51) and a sump (52); said plurality of drainage tubes (51) The water inlets respectively connect a plurality of strip-shaped hydrophilic regions (122) as water inlets of the waste liquid collection device (5), and the liquid collection tanks (52) are disposed on the plurality of drainage tubes (51) Below the water outlet, the drain tube (51) is used to draw excess reaction liquid, and the liquid collection tank (52) is used to collect excess reaction liquid.
The automatic detection system according to claim 1, further comprising a drying device which 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 according to claim 1, further comprising an ultrasonic probe (3) disposed opposite to the strip-shaped hydrophilic region (122) for accelerating the reaction speed of the immunoblotting .
The automatic detection system according to claim 1, further comprising a signal analysis device (4), said signal analysis device (4) being disposed opposite to a surface of said reaction device, said signal analysis device (4) for acquiring Western blot detection signal.
The automatic detection system according to claim 11, wherein said signal analysis means (4) comprises a light source (42) and an image sensor (41); said light source (42) is for emitting light to cause strip-like pro The water region (122) forms a detection signal for the immunoblotting, and the image sensor (41) is used to acquire a detection signal for the immunoblotting.
The automatic detection system according to claim 11, further comprising an alarm device, an output of said signal analysis device (4) being coupled to an input of said alarm device, said alarm device being operative to detect said As a result, an end alert or a failure alert of the immunoblot is obtained.
An immunoblotted film (12), characterized in that the surface of the immunoblot film (12) has a plurality of parallel strip-shaped hydrophilic regions (122) spaced apart by a hydrophobic region (121), the strips being hydrophilic The region (122) has one or more binding regions (122a) having a target antigen for binding to an immunoblotted antibody of interest.
The immunoblot film (12) according to claim 14, wherein the material of the strip-shaped hydrophilic region (122) is nitrocellulose, nylon or polyvinylidene fluoride.
The immunoblotted film (12) according to claim 14, wherein the hydrophobic region (121) has a silane group.
The immunoblot film (12) according to claim 14, wherein the strip-shaped hydrophilic region (122) and the hydrophobic region (121) have a width of from 1 mm to 5 mm.
The immunoblot film (12) according to claim 14, wherein the immunoblotting film (12) is wound into a reel structure in the direction of the strip-shaped hydrophilic region (122).
The immunoblotting film (12) according to claim 18, wherein the reel structure has a diameter of 2 cm to 6 cm and a width of 2 cm to 6 cm.
A method of preparing the immunoblotting film (12) according to claim 14, wherein the target antigen is bound to the hydrophobic membrane to form a binding band of the same amount as the target antigen, and the material of the hydrophobic membrane is Nitrocellulose, nylon or polyvinylidene fluoride;

A hydrophobic region (121) is formed on the hydrophobic film with a hydrophobic coating to separate the hydrophobic film into a plurality of strip-shaped hydrophilic regions (122) while separating the bonding bands into binding regions (122a) such that each strip is hydrophilic Regions (122) all have the same number of binding regions (122a) as the target antigen.
A method of preparing the immunoblotting film (12) according to claim 14, wherein a plurality of strip-shaped hydrophilic films are attached in parallel to the hydrophobic film such that the hydrophobic film has a plurality of strips thereon A plurality of parallel strip-shaped hydrophilic regions (122) formed by the hydrophilic film, the strip-shaped hydrophilic regions (122) being spaced apart by the hydrophobic region (121).
The method according to claim 21, wherein said hydrophobic film surface has a groove that cooperates with a strip-shaped hydrophilic film for fixing a strip-shaped hydrophilic film.
A liquid adding device (2), characterized in that the liquid adding device (2) has one or more water inlets and a plurality of water outlets, and the material of the water outlet is a hydrophilic material to adsorb the reaction liquid. The shape is circular or elliptical, 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.
A liquid adding device (2) according to claim 23, comprising a plurality of liquid adding pipes (21) and a liquid adding pump, the liquid adding pump having a plurality of filling pipes (21) corresponding to each other a water outlet, each water outlet of the liquid adding pump is respectively connected with the water inlets of the plurality of liquid feeding tubes (21) to form a plurality of liquid adding channels, and the water inlet of the liquid adding pump serves as the liquid adding The water inlet of the device (2), the water outlet of the liquid adding pipe (21) serves as the water outlet of the liquid adding device (2).
A liquid filling device (2) according to claim 24, wherein the dosing pump is a syringe pump or a peristaltic pump.
A liquid filling device (2) according to claim 24, wherein said liquid adding device (2) further comprises a fixing rod (23) having a plurality of fixing portions for The plurality of liquid filling tubes (21) are fixed on the same straight line.
A liquid filling device (2) according to claim 24, characterized in that the liquid filling tube (21) is a hydrophilic material.
The liquid adding device (2) according to claim 23, wherein the water inlet of the liquid adding device (2) has a hydrophilic coating, and the hydrophilic coating is polyvinylpyrrolidone or poly N-ethylene. Butyl lactam, poly N-vinylcaprolactam, polyvinyl alcohol, polyvinyl alcohol derivatives, polyurethane or modified polyethylene.