Staining device and biological tissue marking device

Abstract

The present invention provides a staining device and a biological tissue marking device with a simple system, small volume, easy implementation, low cost, and good marking effect. The staining device provided by the present invention is powered on to mark biological tissues, and includes: a staining chamber main body having a receiving cavity; an antibody tank detachably disposed in the receiving cavity, with semi-permeable membranes symmetrically disposed on two sides thereof and communicating with the staining chamber main body for containing an antibody solution as a staining solution; a sample tank detachably disposed in the antibody tank, and a through hole for containing the biological tissue is disposed at a position corresponding to the semi-permeable membrane; and two electrode plates parallel and symmetrically disposed on both sides of the antibody tank, with the lower ends connected to an external power source of the staining chamber main body for energizing the antibody surrounding the biological tissue and marking the biological tissue. Wherein, the molecular cut-off flow rate of the semi-permeable membrane is 6 to 100 kDa, and the material of the antibody tank is an insulating material.

Description

Technical Field

[0001] The present invention relates to the technical field of biological experiments, and particularly to a staining device and a biological tissue marking device. Background Art

[0002] Although staining biological tissues is a common working method in the biological and medical fields, staining thick tissues (with a thickness above the millimeter level) is one of the difficult problems in the field of life sciences. The traditional passive diffusion tissue marking method based on staining molecules (usually antibodies) usually takes several weeks to complete the staining of a thick tissue once. The active diffusion method driven by external forces (such as electric fields, etc.) can accelerate the transport speed of staining molecules in tissues and greatly reduce the staining time. However, existing immunolabeling instruments accelerate the diffusion of antibody molecules in dense tissues based on the principle of random electric field transport. Therefore, the electric field strength needs to be greater than 1151 V / m. To meet this condition, the marking voltage is generally above 100 V, making the device not only complex and large in volume, but also generating a large amount of heat. At a voltage of 100 V, the current is generally above 1 A, and the total power is above 100 W. Therefore, a refrigeration system must be provided, otherwise the temperature of the system will quickly exceed the temperature limit that the sample can withstand (i.e., 37 °C), increasing the cost of the entire immunolabeling instrument. There is an urgent need for a small-sized and low-cost immunolabeling instrument now. Summary of the Invention [[ID=!2]]

[0003] Therefore, in order to overcome the above-mentioned disadvantages of the prior art, a staining device and a biological tissue marking device are provided, which have a simple system, a small volume, are easy to implement, have a low cost, and have a good marking effect.

[0004] To achieve the above object, the present invention provides a staining device that is energized to mark a biological tissue, including: a staining chamber main body having an accommodation cavity; an antibody tank detachably arranged in the accommodation cavity, with semi-permeable membranes symmetrically arranged on two sides and communicating with the staining chamber main body for accommodating an antibody solution as a staining solution; a sample tank detachably arranged in the antibody tank, and a through hole for accommodating the biological tissue is arranged at a position corresponding to the semi-permeable membrane; and two electrode plates arranged in parallel and symmetrically on both sides of the antibody tank, with the lower ends connected to an external power supply of the staining chamber main body for energizing the antibody surrounding the biological tissue and marking the biological tissue. Among them, the molecular cut-off flow rate of the semi-permeable membrane is 6 - 100 kDa, and the material of the antibody tank is an insulating material.

[0005] In one embodiment, a partition for installing the antibody tank is arranged in the accommodation cavity of the staining chamber main body.

[0006] In one embodiment, the electrode plate is a sheet material wound with a conductive wire or a conductive sheet.

[0007] In one embodiment, the electrode plate is a platinum sheet, a gold sheet or a graphite plate.

[0008] In one embodiment, the size range of the semi-permeable membrane is 5mm * 5mm to 50mm * 50mm.

[0009] The present invention also provides a biological tissue marking device, comprising: a staining device for marking biological tissue; and an electrophoresis device electrically connected to the staining device and connected to an external power supply for providing current during marking, wherein the staining device is the above-mentioned staining device.

[0010] In one embodiment, the current control device includes: a housing provided with a display screen for receiving staining data; a staining chamber power supply detachably electrically connected to the staining device for energizing the antibody in the staining device, having a power input port communicating with an external electrophoresis power supply and a current output port connected to the staining device; a control chip electrically connected to the display screen and the current output port for controlling the current value and current direction output from the current output port according to the staining data; and a working power input port communicating with an external working power supply for supplying power to the display screen and the control chip.

[0011] Compared with the prior art, the advantages of the present invention are as follows: Based on the principle of low electric field-assisted antibody molecule diffusion, by applying an external weak electrostatic field (<600V / m), while the antibody molecules freely diffuse, an external force is superimposed to accelerate the diffusion of antibody molecules in dense tissues. Therefore, the working voltage is less than 30V. Moreover, in order to ensure that all current can only pass through biological tissues, the surrounding of the biological tissue is completely blocked with a non-conductive resin material. Under the condition of high resistance of the sample, the working current is only in the mA order of magnitude. Therefore, the power is less than 1W order of magnitude, and the heat generated is almost negligible, without the need to add an additional cooling system. The entire device is simple, small in size, easy to implement, low in cost, and good in marking effect, and can achieve rapid and uniform immunofluorescence marking of thick (thickness above the millimeter order) biological tissues (animals, clinical samples and plants). Description of the Drawings

[0012] Figure 1 is a schematic structural diagram of a biological tissue marking device in an embodiment of the present invention;

[0013] Figure 2 is a schematic structural diagram of a staining device in an embodiment of the present invention;

[0014] Figure 3 is an exploded view of a staining device in an embodiment of the present invention;

[0015] Figure 4It is the staining effect diagram of the biological tissue marking device in the embodiment of the present invention;

[0016] Figure 5 It is the staining effect diagram of the existing device. Detailed implementation manners

[0017] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0018] As Figure 1 shown, in an embodiment of the present invention, the biological tissue marking device 100 includes a staining device 200 and an electrophoresis device 300.

[0019] The staining device 200 is used to fix biological tissues and mark biological tissues. The biological tissues can be animal tissues, clinical sample tissues and plant tissues, with a thickness ranging from the millimeter level to above the centimeter level. They can be samples after clearing treatment or samples without clearing treatment. As Figure 2 and Figure 3 shown, the staining device 200 includes a staining chamber main body 10, an antibody tank 20, a sample tank 30 and an electrode plate 40.

[0020] The staining chamber main body 10 has a receiving cavity. The staining chamber main body 10 has a magnetic base 11 and a cylindrical housing 12. The staining chamber main body 10 can be made by 3D printing or non-metal processing technology, and is made of a non-metal material that is corrosion-resistant and not easily worn, usually including photosensitive resin, plexiglass, glass, plastic, etc. The staining chamber main body can have a circular or square receiving cavity. In this embodiment, the receiving cavity can be a cylinder with a diameter of 40 - 120 mm and a height of 40 - 120 mm. A partition for the antibody tank 20 can be provided in the receiving cavity, and the partition delimits a rectangular area from the receiving cavity. This rectangular area is the electrophoresis immunolabeling area, and the side length can be 20 - 50 mm. The receiving cavity is divided into two parts as buffer zones by the rectangular area. In one embodiment, the receiving cavity is a cylindrical chamber with a diameter of about 78 mm and a height of about 80 mm, and the rectangular area is 30 mm * 36 mm. The cup body design of the arc-shaped receiving cavity is easier for the operator to hold, not easy to drop, and at the same time improves the aesthetics.

[0021] The antibody tank 20 is detachably arranged in the receiving cavity and is used to hold the antibody solution as the staining solution. The antibody tank is a rectangular square tank. An arc-shaped handle is provided on the short side of the rectangular square tank, which can facilitate the operator to take out the antibody tank 20 from the receiving cavity. The material of the antibody tank is an insulating material, and the resistivity of the insulating material is greater than 10 9 , ,

[0021] Ω·m. Through holes that can communicate with the main body of the staining chamber are symmetrically arranged on the sides where the two long sides are located. A semi-permeable membrane is provided on the through holes. The material of the semi-permeable membrane is regenerated fiber or other fiber materials, and the molecular cut-off flow rate of the semi-permeable membrane is 6-100 kDa. The size range of the semi-permeable membrane is 5 mm * 5 mm to 50 mm * 50 mm. In one embodiment, the size of the semi-permeable membrane is 15 mm * 25 mm.

[0022] The sample tank 30 is detachably arranged in the antibody tank 20, and a through hole for accommodating biological tissue is provided at a position corresponding to the semi-permeable membrane. The diameter of the through hole can be 15-60 mm. In one embodiment, the diameter of the through hole is 33 mm. The sample tank 30 is provided with a handle along the long side for easy taking. The biological tissue can be fixed in the through hole by epoxy resin glue, agarose, etc.

[0023] There are two electrode plates 40, which are arranged in parallel and symmetrically on both sides of the antibody tank. The lower end of the electrode plate 40 is connected to an external power supply of the main body of the staining chamber, used to energize the antibody surrounding the biological tissue and label the biological tissue. In one embodiment, the electrode plate 40 is a sheet material wound with a conductive wire or a conductive sheet. The electrode plate 40 can be a metal or non-metal material such as a platinum sheet, a gold sheet or a graphite plate that can be used as an electrode. The electrode plate 40 can also be a metal plate fixed with a specific electrode. The distance between the two electrode plates 40 can be 15-60 mm. In one embodiment, the distance is 33 mm. A small hole opening is provided at a preset position of the main body of the staining chamber, and the lower end of the electrode plate 40 passes through the small hole opening and is electrically connected to a non-wired connector of an external electrophoresis device 300. A waterproof silicone rubber is provided at the small hole opening to ensure that the staining solution does not leak into the circuit.

[0024] The electrophoresis device 300 is electrically connected to the staining device 200 and is used to provide current during labeling. The electrophoresis device 300 includes a housing 301, a staining chamber power supply 302, a control chip 303 and a working power input port 304.

[0025] The housing 301 is provided with a display screen for receiving staining data. The housing 301 can be made of a metal material, a plastic material or a resin material with a certain strength. The housing has a support structure and functions to protect the internal circuit. The front panel of the housing 301 can be set at an acute angle to the horizontal plane. In one embodiment, the front panel is set to be inclined 30° to the horizontal plane, which can facilitate the operator to read the numbers placed on the front panel and input operations. The size range of the housing 301 can be: length 10-30 cm, width 10-30 cm, height 2-10 cm. In one embodiment, the overall size is length × width × height: 12 * 13 * 2.5 cm. This size is small and light, facilitating daily use in conventional biological laboratories and clinical test laboratories.

[0026] The display screen may include membrane keys, a digital display, and a control panel, thereby realizing the interactive function between the instrument operator and the staining instrument. The membrane keys are keys with input functions processed from materials such as plastic or metal. The digital display shows the key parameters of the system, including the running time, remaining time, etc. Various parameters related to the staining data can be input through the keys to control the operation and stop of the instrument. The control panel is fixed on the front panel of the housing 301 with a certain inclination angle, facilitating the operator to view and operate in a standing or sitting upright state.

[0027] The display screen may also include status indicators. The status indicators can indicate the current state of the instrument, including running and not running, etc. In the running state, the numbers on the display screen will enter a countdown, and in cases where the remaining time is less than 1 - 5 minutes or there is an unexpected stop, a prompt sound will be emitted to remind the operator, so as to perform corresponding operations to ensure the smooth progress of the experiment. The membrane keys, digital display, status indicators, and control panel are electrically connected.

[0028] The power supply 302 of the staining chamber is connected to an external power supply, having a power input port 3021 provided on the rear panel of the housing 301 and a current output port 3022 connected to the staining device.

[0029] The power input port 3021 can be connected to a constant current source or to a 220V AC circuit through a power adapter, and is equipped with a power switch. All connectors are firmly connected, and an insulating protective cover is provided to effectively prevent electric leakage.

[0030] The current output port 3022 is electrically connected to the staining device. The current output port 3022 is provided at the top of the housing 301 and is connected to the connection joint at the bottom of the staining device 200. The current output port 3022 can adopt any one of the connection methods such as magnetic connectors, banana plugs, socket heads, etc. to realize the separation and connection of the staining device 200 and the electrophoresis device 300. The connector conduction can stably maintain a current of the ampere level, maintain at least tens of thousands of plug - and - unplug times without affecting the service life of the instrument.

[0031] The control chip 303 is electrically connected to the display screen, the power input port 3021, and the current output port 3022, and controls the current value and current direction output by the current output port 3022 according to the staining data. The control chip 303 is fixed on the bottom plate inside the housing 301. In this embodiment, the control chip 303 is a printed circuit chip board (PCB circuit board). Through this PCB circuit board, automatic current control of the staining process can be achieved. The total energization time can be input through the display screen according to experimental requirements. The control chip 303 automatically controls the electrophoresis power supply to provide a constant current for the staining device 200 according to the total time. When the running time reaches exactly half of the total time, the control chip 303 automatically reverses the current and maintains a constant current until the experiment ends. By reversing the current, two immunolabelings of the experimental sample are achieved, ensuring the uniformity of immunolabeling. The control chip 303 can memorize the time input by the operator last time and display it on the display screen, which is convenient for the operator to repeat the experiment or perform other operations. During the experiment, the operator can control the operation, pause, continuation, and stop of the instrument through the buttons on the display screen. Before running, the control chip 303 will detect whether the circuit connection is normal. If the staining device is not placed correctly, resulting in an abnormal circuit connection, it will automatically remind the operator to re-place the staining device.

[0032] The working power input port 304 is connected to an external working power supply and is used to supply power to the display screen and the control chip 303 of the housing 301. The working power input port 304 can be connected to a power adapter to a 220V AC power supply, and then provide a stable 12 - 24V working voltage.

[0033] The usage process of the staining device 200 is as follows:

[0034] Fix the experimental sample (thick biological tissue) in the circular cavity of the sample tank 30 through a fixative; then place the sample tank 30 at the corresponding position of the antibody tank 20; after that, the antibody diluent (dilution ratio 1:200 - 1:10000 times) can be added to both sides of the sample tank 30 in the antibody tank 20 until it covers the semi-permeable membrane of the antibody tank 20; place the antibody tank 20 at the corresponding position of the staining chamber main body 10, and add staining solution to the remaining space of the staining chamber until it covers the electrode plate.

[0035] The usage process of the biological tissue labeling device is as follows:

[0036] Place the staining device 200 on the top of the electrophoresis device 300; connect the external power supply to the working power input port 304 on the rear panel of the control box, and turn on the instrument power switch to make the instrument in a standby state for operation;

[0037] Input the total duration of the experiment through the display screen; after setting is completed, click the run button, and the control chip 303 will automatically calculate the time before and after the current reverses, switch the status indicator light, and conduct the circuit in the forward direction. Wait until the running time in one direction (defined as the forward direction) ends, and then automatically switch to reverse conduction until the experiment ends;

[0038] There is a prompt sound at the end of the experiment. The operator can take out the sample, or replace the kit for cleaning, or replace the antibody for re-immunolabeling.

[0039] The above-mentioned staining device and biological tissue labeling device are based on the principle of low-electric-field-assisted antibody molecule diffusion. Through an externally applied weak electrostatic field (<600 V / m), while the antibody molecules freely diffuse, an external force is superimposed to accelerate the diffusion of antibody molecules in dense tissues. Therefore, the working voltage is less than 30 V. Moreover, in order to ensure that all currents can only pass through biological tissues, the surrounding of the biological tissue is completely blocked with non-conductive resin materials. Under the condition of high resistance of the sample, the working current is only in the mA range. Therefore, the power will not exceed 1 W, and the generated heat can be almost ignored, and there is no need to add an additional cooling system. The whole device is simple, small in size, easy to implement, low in cost, and good in labeling effect, and can achieve rapid and uniform immunofluorescent labeling of thick (with a thickness of more than millimeter level) biological tissues (animals, clinical samples, and plants).

[0040] In one embodiment, the main body of the staining chamber is further provided with a cover, which can effectively prevent the staining solution from overflowing. There is a rectangular liquid inlet and outlet with a side length of 5 - 40 mm on the cover, and this rectangular liquid inlet and outlet can facilitate the operator to add liquid and clean. In one embodiment, the size of this rectangular liquid inlet and outlet can be 15 mm * 25 mm.

[0041] For example, an Anti-Histone H3 antibody with Alexa-647 fluorescent molecules can be used to stain and label a 2-mm-thick cleared brain tissue, as follows:

[0042] First, use the standard CLARITY clearing method to clear a 2-mm-thick mouse brain (C57BL / 6) tissue;

[0043] Then fix the sample in the sample tank 30 with epoxy resin glue, and place the sample tank 30 at the corresponding position of the antibody tank 20;

[0044] Add the Anti-histone H3 antibody solution diluted at 1:200 to the antibody tank 20 until it covers the semi-permeable membrane of the antibody tank 20;

[0045] Place the antibody tank 20 at the corresponding position of the main body 10 of the staining chamber, and add 100 mL of staining solution to the remaining space of the staining chamber to cover the electrode plate;

[0046] Place the staining device 200 on top of the electrophoresis device 300;

[0047] Turn on the power of the staining device, input the staining time as 3 hours, and start the staining device;

[0048] After the staining is completed, take out the sample, place it in a sample tank dedicated for elution, and replace the antibody solution with a buffer dedicated for elution;

[0049] Put the sample into the staining chamber, turn on the power and input the time as 3 hours, start the staining device, and realize the elution of the antibody.

[0050] Figure 4 It is to take the cross-sectional view of the brain tissue section stained by using the biological tissue marking device of this embodiment through a confocal fluorescence microscope. During this experiment, the staining time was 3 hours, the elution time was 3 hours, the voltage output was 20V, the current output was 20mA, the power output was only 0.4W, the actual usage amount (concentration × volume) of the antibody was only 0.6 μg, and the temperature rise of the system during the whole staining process did not exceed 1°C. As Figure 4 shown, the antibody staining of the cross-section of the 2-mm brain slice is very uniform.

[0051] Under the same conditions, Figure 5 It is the fluorescence image of the cross-section of the brain slice after staining the mouse brain tissue (2-mm thickness) with a staining device using the passive staining method at the same staining time and antibody concentration. Figure 4 and Figure 5 The white scale bar of Figure 5 is 200 μm. As Figure 4 shown, the staining device only labels a very thin area on the surface (<200 μm), and to achieve the staining effect as

[0052] it is necessary to extend the immunolabeling time by more than 10 times.

[0053] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. The above is only the preferred implementation manner of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can still be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A staining device that is powered on to label biological tissues, characterized in that, Comprising: A main body of a staining chamber, having a receiving cavity, in which a partition is provided, and the partition delimits a rectangular area in the receiving cavity, and this rectangular area is an electrophoresis immunolabeling area; An antibody tank, detachably provided in the rectangular area of the receiving cavity. The antibody tank is a rectangular square tank, and an arc-shaped handle is provided on the short side of the rectangular square tank for taking out the antibody tank from the receiving cavity; Semi-permeable membranes communicating with the main body of the staining chamber are symmetrically arranged on two sides for containing an antibody solution as a staining solution; A sample tank, detachably provided in the antibody tank, a through hole for accommodating the biological tissue is provided at a position corresponding to the semi-permeable membrane, and a handle is provided along the long side of the sample tank for easy taking; And Two electrode plates, arranged in parallel and symmetrically on both sides of the antibody tank, the lower ends of which are connected to an external power supply of the main body of the staining chamber, for energizing the antibody surrounding the biological tissue and labeling the biological tissue. A small hole opening is provided at a preset position of the main body of the staining chamber, and the lower ends of the electrode plates pass through the small hole opening and are electrically connected to a non-wired connector of an external electrophoresis device, and a waterproof silicone rubber is provided at the small hole opening; Among them, the molecular cut-off flow rate of the semi-permeable membrane is 6 to 100 kDa, the material of the antibody groove is an insulating material, the resistivity of the insulating material is greater than 10 9 Ω·m, and the thickness of the biological tissue is above the millimeter level.

2. The dyeing device according to claim 1, wherein The electrode plate is a sheet material wound with a conductive wire or a conductive sheet.

3. The dyeing device according to claim 1, wherein The electrode plate is a platinum sheet or a gold sheet.

4. The dyeing device according to claim 1, characterized in that, The size range of the semi-permeable membrane is 5mm*5mm~50mm*50mm.

5. A biological tissue marking device, characterized in that, Comprising: A staining device for labeling biological tissues; And An electrophoresis device, electrically connected to the staining device and connected to an external power supply, for providing current during labeling, wherein, the staining device is the staining device according to any one of claims 1 to 4.

6. The biological tissue marking device according to claim 5, wherein, The electrophoresis device includes: A housing, provided with a display screen for receiving staining data; A staining chamber power supply, detachably electrically connected to the staining device, for energizing the antibody in the staining device, having a power input port communicating with an external electrophoresis power supply and a current output port connected to the staining device; A control chip, electrically connected to the display screen and the current output port, for controlling the current value and current direction output by the current output port according to the staining data; and A working power input port, communicating with an external working power supply, for supplying power to the display screen and the control chip.

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