An automated high-throughput immunostaining sample preparation system and methods of use thereof

Abstract

The application relates to the technical field of immunofluorescence imaging, in particular to an automatic high-throughput immunostaining sample preparation system and a use method thereof, which comprises a host computer, a plurality of adapter groups, a peristaltic pump a, a peristaltic pump b, a plurality of communication modules, a channel switching valve a, a channel switching valve b and a channel switching valve c; the host computer is connected to the peristaltic pump a, the peristaltic pump b, the channel switching valve a, the channel switching valve b and the channel switching valve c through the communication modules; the peristaltic pump b is connected with a waste liquid bottle; the peristaltic pump a is connected to a sample supply assembly through the channel switching valve c; the sample supply assembly is loaded with a sample preparation liquid; the plurality of adapter groups are arranged between the channel switching valve a and the channel switching valve b; the channel switching valve a is connected to the peristaltic pump a; the channel switching valve b is connected to the peristaltic pump b; and the channel switching valve a is connected to the waste liquid bottle. The application improves the throughput of automatic immunostaining sample preparation, realizes automatic high-throughput sample preparation, and improves work efficiency.

Description

Technical Field

[0001] This invention relates to the field of immunofluorescence imaging technology, and in particular to an automated high-throughput immunostaining sample preparation system and its usage method. Background Technology

[0002] Existing patent CN118294651A discloses an automated sample preparation machine and method. The automated sample preparation machine includes: an adapter; a syringe outlet for each syringe adapted to communicate with a corresponding liquid injection port of the adapter; a sample preparation drive assembly adapted to drive multiple syringes to inject the required reagent solution into the confocal dish adapter base of the adapter; an inlet for a waste discharge assembly connected to the drain port of the adapter, adapted to sequentially discharge waste liquid generated during sample preparation; and a controller configured to determine the sample preparation sequence when multiple syringes are drawing in reagent solution, and to control the sample preparation drive assembly and waste discharge assembly according to the sample preparation sequence, so that the multiple syringes work alternately with the waste discharge assembly. The automated sample preparation machine involved in this patent can only prepare one sample per work cycle. When the number of samples required for the experiment increases, the time required is multiplied accordingly, resulting in a significant waste of time and labor. Therefore, in practical applications, this single-throughput automated sample preparation machine exposes certain limitations, restricting its application and promotion in a wider range of fields. Summary of the Invention

[0003] This invention aims to at least solve one of the technical problems existing in related technologies. To this end, this invention provides an automated high-throughput immunostaining sample preparation system and its method of use, solving the low-throughput problem in the prior art and enabling the preparation of multiple samples in a single work cycle. During sample preparation, no laboratory personnel are required, and only a small amount of simple reagent preparation is needed, greatly saving time and labor costs, and significantly increasing the number of samples prepared in a single experiment.

[0004] This invention provides an automated high-throughput immunostaining sample preparation system, including a host computer, several sets of adapters, peristaltic pump a, peristaltic pump b, several communication modules, channel switching valve a, channel switching valve b, and channel switching valve c; The host computer is connected to peristaltic pump a and peristaltic pump b. Peristaltic pump b is connected to a waste liquid bottle. Peristaltic pump a is connected to a sample supply assembly via a channel switching valve c. The sample supply assembly is loaded with sample preparation solution. Several sets of adapters are disposed between channel switching valve a and channel switching valve b. Channel switching valve a is connected to peristaltic pump a, channel switching valve b is connected to peristaltic pump b, and channel switching valve a is connected to the waste liquid bottle. Communication modules are provided between the host computer and the peristaltic pump a, between the host computer and the peristaltic pump b, between the host computer and the channel switching valve a, between the host computer and the channel switching valve b, and between the host computer and the channel switching valve c.

[0005] A further improvement of the automated high-throughput immunostaining sample preparation system of the present invention is that the adapter is a multi-well plate adapter, the multi-well plate adapter comprising: A perforated plate having multiple perforations and slots; The cover plate has two insertion holes corresponding to each of the slots, and the cover plate has a mounting groove. The cover plate is disposed on the perforated plate. The pagoda connector has a first hole and a second hole, which are concentrically arranged. The inner diameter of the first hole is larger than the inner diameter of the second hole. The pagoda connector has an external thread. The top of the pagoda connector is connected to the channel switching valve a or the channel switching valve b through a pipe. A steel needle, wherein the steel needle is hollow and straight, and the first end of the steel needle is at an angle and the second end is at a flat angle; A limiting component is installed on the cover plate and is used to fix the position of the pagoda connector.

[0006] A further improvement of the automated high-throughput immunostaining sample preparation system of the present invention is that the second end of the steel needle is inserted and connected to the first hole of the pagoda connector; the limiting component includes a spring and a nut, the spring is sleeved on the pagoda connector, the spring is disposed between the pagoda connector and the cover plate, and the axial elastic force generated by the spring enables the first end of the steel needle to contact the bottom of the slot of the multi-well plate.

[0007] A further improvement of the automated high-throughput immunostaining sample preparation system of the present invention is that the cover plate has two insertion holes for each of the slots, the pagoda connector passes through the insertion holes of the cover plate, and the external thread of the pagoda connector is connected to the nut so that the pagoda connector is fixed to the insertion holes of the cover plate. The top of one of the pagoda connectors passing through the insertion hole is connected to the channel switching valve a through a pipe, and the top of the other pagoda connector is connected to the channel switching valve b through a pipe.

[0008] A further improvement of the automated high-throughput immunostaining sample preparation system of the present invention is that the adapter is a confocal dish adapter, the confocal dish adapter comprising: A confocal dish adapter base is provided, in which a confocal dish is placed. The confocal dish is covered with a limiting ring, and a first through hole is formed in the middle of the limiting ring. A boss is formed at the end of the limiting ring corresponding to the position of the first through hole, and a first sealing ring is fitted on the boss. The cover is placed on the confocal dish adapter base. The top of the cover has two first mounting slots. The cover has a second through hole. The top of the second through hole is connected to the first mounting slot. The cover has two second mounting slots. The two second mounting slots and the two first mounting slots are arranged in a one-to-one correspondence. The first end of the second mounting slot is connected to the first mounting slot. Two capillaries, each L-shaped, each including a first section and a second section, the first section passing through the second through hole, the bottom end of the first section abutting against the confocal dish, the second section placed in the first mounting groove, one of the capillaries being connected to the channel switching valve a via a pipe, and the other capillary being connected to the channel switching valve b via a pipe; A pressure limiting assembly is installed on the top of the cover body to limit the position of the second pipe segment. The pressure limiting assembly includes a spring plate, the first end of which is connected to the bottom of the second mounting groove and located near the second end of the second mounting groove, and the second end of which abuts against the cover body and covers the first mounting groove.

[0009] A further improvement of the automated high-throughput immunostaining sample preparation system of the present invention is that the first end of the spring plate is connected to the second mounting groove by a fixing screw, the second mounting groove is provided with a threaded hole near the second end, the fixing screw is screwed into the threaded hole, and the first end of the spring plate is formed with a U-shaped end, and the fixing screw is connected to the U-shaped end; An internal thread section is formed at the top of the inner wall of the confocal dish adapter base, and an external thread section is formed at the bottom of the outer wall of the cover. The external thread section and the internal thread section are screwed together. The side of the limiting ring forms a limiting groove, and the inner sidewall of the confocal dish adapter base forms a first step, with the limiting groove placed on the first step.

[0010] A further improvement of the automated high-throughput immunostaining sample preparation system of the present invention is that the adapter is a tissue section adapter, the tissue section adapter comprising: A tissue slide adapter base has a limiting hole for placing a sample carrier. An observation hole is provided at the bottom of the tissue slide adapter base. A limiting gasket is provided on the sample carrier, and a second sealing ring is fitted on the limiting gasket. A cover body is provided on the tissue slide adapter base. The top of the cover body has two first mounting slots. The cover body has a second through hole. The top end of the second through hole is connected to the first mounting slot. The cover body has two second mounting slots. The two second mounting slots and the two first mounting slots are arranged in a one-to-one correspondence. The first end of the second mounting slot is connected to the first mounting slot. Two capillaries, each L-shaped, each including a first section and a second section, the first section passing through the second through hole, the bottom end of the first section abutting against the sample carrier, the second section placed in the first mounting groove, one of the capillaries being connected to the channel switching valve a via a pipe, and the other capillary being connected to the channel switching valve b via a pipe; A pressure limiting assembly is installed on the top of the cover body to limit the position of the second pipe segment. The pressure limiting assembly includes a spring plate, the first end of which is connected to the bottom of the second mounting groove and located near the second end of the second mounting groove, and the second end of which abuts against the cover body and covers the first mounting groove.

[0011] A further improvement of the automated high-throughput immunostaining sample preparation system of the present invention is that the first end of the spring plate is connected to the second mounting groove by a fixing screw, the second mounting groove is provided with a threaded hole near the second end, the fixing screw is screwed into the threaded hole, and the first end of the spring plate is formed with a U-shaped end, and the fixing screw is connected to the U-shaped end; An internal thread section is formed at the top of the inner wall of the limiting hole, and an external thread section is formed at the bottom of the outer wall of the cover. The external thread section and the internal thread section are screwed together.

[0012] The present invention discloses a method for using an automated high-throughput immunostaining sample preparation system. The method, performed using the automated high-throughput immunostaining sample preparation system described above, includes the following steps: Multiple sample preparation solutions are loaded into the sample supply assembly. These solutions include washing solution, fixative solution, PBS solution, blocking solution, primary antibody solution, WB solution, and secondary antibody solution. Several channels of channel switching valve c correspond to one sample preparation solution. Each channel of channel switching valve a and channel switching valve b corresponds to an adapter. Sample preparation parameters are set in the host computer. The host computer starts the peristaltic pump a through the communication module. The host computer opens the corresponding channel positions of channel switching valve a and channel switching valve c through the communication module so that the peristaltic pump a transfers the sample preparation liquid corresponding to the sample supply component into the corresponding adapter. The host computer starts the peristaltic pump b through the communication module, and opens the corresponding channel position of the corresponding channel switching valve b through the communication module, so that the peristaltic pump b transfers the liquid in the corresponding adapter into the waste liquid bottle.

[0013] The automated sample preparation system of this invention uses a host computer to control peristaltic pumps a, b, channel switching valves a, b, and c via a communication module. This precise control of multiple adapters improves the automation level of sample preparation, enabling fully automated operation, avoiding sample contamination, and increasing the success rate of sample culture. This automated sample preparation system can prepare multiple samples in a single work cycle. During sample preparation, no human presence is required, and only a small amount of simple reagent preparation is needed, significantly saving time and labor costs, and greatly increasing the number of samples prepared in a single experiment.

[0014] The multi-well plate adapter of the present invention facilitates simultaneous sample preparation at multiple locations via a multi-well plate. A pagoda connector facilitates the entry of sample solution into the multi-well plate. The inner diameter of the first hole of the pagoda connector is not equal to the inner diameter of the second hole. The first end of the steel needle is designed with an angled bevel for easy piercing of the sample container, while the second end is designed with a flat angle for easy connection with the pagoda connector. The limiting component includes a nut and a spring. Under the action of the spring, the nut can tightly press the pagoda connector to prevent it from loosening during use.

[0015] The confocal dish adapter of this invention employs an L-shaped capillary tube, with the bottom of the first tube section adjacent to the bottom of the confocal dish, enabling precise liquid delivery and making formulation introduction and cleaning solution entry and exit more efficient and accurate. The first mounting groove, second through-hole, and pressure limiting component of the cap body work together to stabilize the capillary position, improving the reliability and repeatability of the device operation. In experimental operation, liquid handling is convenient and efficient, reducing operational difficulty and facilitating the standardization of experimental procedures. Precise liquid control reduces experimental errors and improves the reliability of results, providing more efficient and precise tool support for confocal-related research such as cell observation and drug screening, thus contributing to improved research efficiency and quality. By setting a second mounting groove in the cap body, the pressure limiting component presses against the capillary, improving the stability of the capillary.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a connection diagram of an automated high-throughput immunostaining sample preparation system provided by the present invention.

[0019] Figure 2 This is a schematic diagram of an automated high-throughput immunostaining sample preparation system provided by the present invention.

[0020] Figure 3 This is an exploded view of the perforated plate adapter in Embodiment 1 of the present invention.

[0021] Figure 4 This is a schematic diagram of the perforated plate adapter in Embodiment 1 of the present invention.

[0022] Figure 5 This is a schematic diagram of the perforated plate in the perforated plate adapter of Embodiment 1 of the present invention.

[0023] Figure 6 This is a schematic diagram of the cover plate in the perforated plate adapter of Embodiment 1 of the present invention.

[0024] Figure 7 This is a schematic diagram of the steel needle in the perforated plate adapter in Embodiment 1 of the present invention.

[0025] Figure 8 This is a schematic diagram of the pagoda connector in the multi-hole plate adapter of Embodiment 1 of the present invention.

[0026] Figure 9 This is an exploded view of the co-focusing dish adapter according to Embodiment 2 of the present invention.

[0027] Figure 10 This is a schematic diagram of the confocal adapter in Embodiment 2 of the present invention.

[0028] Figure 11 This is a schematic diagram of the cover in the cofocusing adapter of Embodiment 2 of the present invention.

[0029] Figure 12 This is a schematic diagram of the cover in the cofocusing adapter of Embodiment 2 of the present invention.

[0030] Figure 13 This is an exploded view of the tissue slice adapter in Embodiment 3 of the present invention.

[0031] Figure 14 This is a schematic diagram of the tissue slice adapter in Embodiment 3 of the present invention.

[0032] Figure label: 1. Host computer; 2. Communication module; 3. Centrifuge tube; 4. Channel switching valve c; 5. Peristaltic pump a; 6. Channel switching valve a; 7. Multi-hole plate adapter; 8. Channel switching valve b; 9. Peristaltic pump b; 10. Waste liquid bottle; 11. USB extender; 101. Multi-hole plate; 1011. Groove; 102. Cover plate; 1021. Socket; 1022. Mounting groove; 103. Steel needle; 1031. Angled; 1032. Flat angle; 104. Pagoda connector; 1041. First hole; 1042. Second hole; 1043. External thread; 105. Spring; 106. Nut; 201. Confocal dish adapter base; 2011. Internal groove thread; 2012. Third step; 201 3. Fourth step; 2014. Observation through hole; 202. Confocal dish; 203. First sealing ring; 204. Limiting ring; 2041. First through hole; 2042. Limiting groove; 205. Cover; 2051. External thread section; 2052. Second mounting groove; 2053. First mounting groove; 2054. Threaded hole; 2055. Second through hole; 2056. Pressure groove; 206. Capillary tube; 207. Fixing screw; 208. Spring pressure plate; 301. Sample carrier; 302. Tissue slide adapter base; 3021. Limiting hole; 3022. Observation hole; 3023. Internal thread section; 303. Second sealing ring; 304. Limiting washer; 3041. Third through hole. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention but should not be used to limit the scope of this invention.

[0034] The following is combined with Figure 1 and Figure 2This invention describes an automated high-throughput immunostaining sample preparation system, comprising a host computer, several sets of adapters, peristaltic pumps a5 and b9, several communication modules, channel switching valves a6, b8, and c4. The host computer is connected to peristaltic pumps a5 and b9. Peristaltic pump b9 is connected to a waste liquid bottle. Peristaltic pump a5 is connected to a sample supply assembly via channel switching valve c4. The sample supply assembly contains sample preparation solution. Several sets of adapters are disposed between channel switching valves a6 and b8. Channel switching valve a6 is connected to peristaltic pump a5, channel switching valve b8 is connected to peristaltic pump b9, and channel switching valve a6 is connected to the waste liquid bottle. Communication modules are provided between the host computer and peristaltic pump a5, between the host computer and peristaltic pump b9, between the host computer and channel switching valve a6, between the host computer and channel switching valve b8, and between the host computer and channel switching valve c4.

[0035] Preferably, the communication module has 5 groups, namely communication module a, communication module b, communication module c, communication module d, and communication module e. Communication module a is located between the host computer and peristaltic pump a5, communication module b is located between the host computer and peristaltic pump b9, communication module d is located between the host computer and channel switching valve a6, communication module e is located between the host computer and channel switching valve b8, and communication module c is located between the host computer and channel switching valve c4.

[0036] Preferably, the host computer, acting as the control core, sends commands to the peristaltic pump a5 via communication module a, controlling it to draw sample preparation solution from the sample supply component and deliver it to the adapter area via channel switching valve c4. Simultaneously, the host computer controls the peristaltic pump b9 via communication module b, allowing it to draw or discharge waste liquid from the waste bottle according to experimental needs, maintaining liquid balance within the system. During sample preparation, channel switching valves a6 and b8, under the control of the host computer, are precisely switched via communication modules d and e, ensuring that the sample preparation solution flows through different adapters along a preset path to complete the immunostaining step. Furthermore, the flexible adjustment of channel switching valve c4 allows the system to adapt to the supply needs of various sample preparation solutions, improving experimental flexibility and efficiency. Throughout the process, the collaborative work between components is achieved through real-time data interaction between the host computer and each communication module, ensuring high throughput and automated operation of the system.

[0037] Preferably, the sample supply component consists of several centrifuge tubes, which can be selected in various sizes, such as 0.2mL, 0.5mL, 1.5mL, 2mL, 5mL, 15mL, and 50mL.

[0038] Better, such as Figure 2As shown, in a specific sample preparation method, there are 7 centrifuge tubes, of which 4 centrifuge tubes are 15mL and 3 centrifuge tubes are 50mL; channel switching valves a6 and b8 each have 1 common channel position and 12 channels, and channel switching valve c4 has 1 common channel position and 8 channels; there are 11 confocal dish adapters, which are located between channels 1 to 11 of channel switching valves a6 and b8, respectively; channel 12 of channel switching valve a is connected to the waste bottle; peristaltic pump a5 is connected to the common channel position of channel switching valves a and c4; peristaltic pump b9 is connected to the common channel position of channel switching valve b8; 7 centrifuge tubes are connected to channels 1 to 7 of channel switching valve c4, and channel 8 is connected to an air device (providing clean air).

[0039] like Figure 2 As shown, four 15 mL centrifuge tubes contained fixative solution, blocking solution, primary antibody solution, and secondary antibody solution, respectively, and three 50 mL centrifuge tubes contained PBS solution (phosphate buffer), WB solution, and washing solution, respectively.

[0040] Example 1: As Figures 3 to 8 As shown, the adapter is a perforated board adapter, which includes: A perforated plate 101, wherein a plurality of holes and grooves 1011 are formed in the perforated plate 101; The cover plate 102 has two insertion holes 1021 for each of the slots 1011, and the cover plate 102 is provided with an installation groove 1022. The cover plate 102 is disposed on the perforated plate 101. The pagoda connector 104 has a first hole 1041 and a second hole 1042, which are concentrically arranged. The inner diameter of the first hole 1041 is larger than the inner diameter of the second hole 1042. The pagoda connector 104 has an external thread 1043. The top of the pagoda connector 104 is connected to the channel switching valve a or the channel switching valve b through a pipe. The pagoda connector 104 passes through the insertion hole 1021 of the cover plate 102. The steel needle 103 is hollow and straight. The first end of the steel needle is at an angle 1031 and the second end is at a flat angle 1032. The second end of the steel needle 103 is inserted and connected to the first hole 1041 of the pagoda connector 104. A limiting component is installed on the cover plate 102 to fix the position of the pagoda connector 104. The limiting component includes a spring 105 and a nut 106. The spring 105 is sleeved on the pagoda connector 104 and is disposed between the pagoda connector 104 and the cover plate 102. The axial elastic force generated by the spring 105 allows the first end of the steel needle 103 to contact the bottom of the slot 1011 of the perforated plate 101. The external thread 1043 of the pagoda connector 104 is connected to the nut 106 so that the pagoda connector 104 is fixed to the insertion hole 1021 of the cover plate 102.

[0041] Preferably, the second end of the steel needle 103 is inserted through the first hole 1041 of the pagoda connector 104 and then glued together. The second end of the steel needle 103 is close to the bottom of the first hole 1041 of the pagoda connector 104, which reduces the dead volume of the liquid in the pagoda connector 104 and reduces the error of the injected liquid.

[0042] Better, such as Figures 3 to 8 As shown, by the bevel of the first end of the steel needle 103 contacting the bottom of the slot 1011 of the multi-well plate 101, the peristaltic pump b9 can ensure that most of the waste liquid is extracted from the corresponding slot 1011 of the multi-well plate 101, thereby avoiding contamination between reagents and improving the accuracy of sample preparation. In terms of experimental operation, liquid handling is convenient and efficient, reducing the difficulty of operation, facilitating the standardization of experimental procedures, and precise liquid control reduces experimental errors and improves the reliability of results. It can provide more efficient and accurate tool support for cell observation, drug screening and other related research, helping to improve scientific research efficiency and quality.

[0043] Specifically, such as Figures 3 to 8 As shown, the pagoda connector 104 passes through the insertion hole 1021 of the cover plate 102. The nut 106 of the limiting component is connected to the external thread 1043 of the pagoda connector 104, ensuring that the pagoda connector 104 is fixed to the insertion hole 1021 of the cover plate 102. This connection method is not only stable and reliable, but also facilitates the disassembly and replacement of the pagoda connector 104. When it is necessary to adjust the pressure of the spring 105 or perform maintenance, simply tighten the nut 106 to easily adjust the pressure of the spring 105 or remove the spring 105, making the operation very convenient. Furthermore, because the reagent contains a salt solution, the materials of the spring 105, cover plate 102, steel needle 103, and pagoda connector are carefully selected to have high strength and corrosion resistance, maintaining stable performance during long-term use and further ensuring the normal operation of the multi-hole plate adapter.

[0044] Better, such as Figures 3 to 8As shown, the pagoda connector 104 is screwed into the nut 106. This threaded connection can withstand greater external forces. Even when the perforated plate adapter is subjected to vibration or impact, the spring 105 will not loosen or fall off, thereby maintaining the stability of the perforated plate adapter.

[0045] Better, such as Figures 3 to 8 As shown, the pagoda connector 104 is close to the side wall of the insertion hole 1021 of the cover plate 102, which ensures that the flow rate of the fluid will not damage the sample when injecting reagent liquid or extracting waste liquid, thus ensuring the reliability and repeatability of sample preparation.

[0046] Better, such as Figures 3 to 8 As shown, the top of the pagoda connector 104 forms an injection port for easy connection to channel switching valve a or channel switching valve b. The cover plate 102 has two insertion holes 1021 corresponding to each of the slots 1011. The pagoda connector 104 passes through the insertion hole 1021 of the cover plate 102. The external thread of the pagoda connector 104 is connected to the nut so that the pagoda connector 104 is fixed to the insertion hole 1021 of the cover plate 102. The top of one of the pagoda connectors passing through the insertion hole is connected to the channel switching valve a through a pipe, and the top of the other pagoda connector is connected to the channel switching valve b through a pipe.

[0047] Example 2, as Figures 9 to 12As shown, the adapter is a confocal dish adapter, including: a confocal dish adapter base 201, in which a confocal dish 202 is placed, and the confocal dish 202 is covered by a limiting ring 204; a first through hole 2041 is formed in the middle of the limiting ring 204, and a boss is formed at the end of the limiting ring 204 corresponding to the channel position, and a first sealing ring 203 is fitted on the boss; a cover 205, which covers the confocal dish adapter base 201, and has two first mounting grooves 2053 on its top, and a second through hole 2055, the top of which communicates with the first mounting grooves 2053; and two second mounting grooves 2052, which are arranged one-to-one with the two first mounting grooves 2053. The first end of the groove 2052 is connected to the first mounting groove 2053; two capillaries 206 are L-shaped and include a first section and a second section. The first section passes through the second through hole 2055, and the bottom end of the first section abuts against the confocal dish 202. The second section is placed in the first mounting groove 2053. The capillaries 206 are connected to the channel switching valve a and the channel switching valve b through pipes. One capillary 206 can be used as an injection port, and the other capillary 206 can be used as a waste aspiration port. The bottom end of the first section of the capillary 206 is close to the bottom of the confocal dish 202, which facilitates the entry of the sample preparation solution into the confocal dish 202 and the entry or aspiration of the cleaning solution into or out of the confocal dish 202; a pressure limiting component is installed on the top of the cover 205 and is used to limit the position of the second section.

[0048] Better, such as Figures 9 to 12 As shown, two L-shaped capillaries 206 are used as the injection port and the waste aspiration port, respectively. Channel switching valve a is connected to the injection port and channel switching valve b is connected to the waste aspiration port. The bottom of the first tube section is close to the bottom of the confocal dish 202 to achieve precise liquid delivery, making the introduction of sample preparation liquid and the entry and exit of cleaning liquid more efficient and accurate. The first mounting groove 2053 and the second through hole 2055 of the cover 205 cooperate with the pressure limiting component to stabilize the position of the capillary 206 and improve the reliability and repeatability of the device operation.

[0049] Specifically, such as Figures 9 to 12 As shown, the pressure limiting assembly includes a spring plate 208. The first end of the spring plate 208 is connected to the bottom of the second mounting groove 2052 and is located near the second end of the second mounting groove 2052. The second end of the spring plate 208 abuts against the cover 205 and covers the first mounting groove 2053.

[0050] Better, such as Figures 9 to 12 As shown, the spring clamp 208 is made of a highly elastic metal material, capable of elastic deformation under external force. When the capillary 206 is placed in the first mounting groove 2053, the spring clamp 208 uses its own elasticity to firmly hold the capillary 206 in place, effectively limiting its horizontal movement and ensuring its stable position during the experiment. The dimensions of the two second mounting grooves 2052 are matched with the first mounting groove 2053, and their depths are precisely designed to ensure that the spring clamp 208 has sufficient mounting space while also providing appropriate pressure on the capillary 206, significantly improving the stability and accuracy of the confocal dish adapter during use.

[0051] Specifically, such as Figures 9 to 12 As shown, the first end of the spring pressure plate 208 is connected to the second mounting groove 2052 via a fixing screw 207. This connection method is not only stable and reliable, but also facilitates the disassembly and replacement of the spring pressure plate 208. When it is necessary to adjust the pressure of the spring pressure plate 208 or to perform maintenance, the spring pressure plate 208 can be easily removed simply by unscrewing the fixing screw 207, making the operation very convenient. Furthermore, the fixing screw 207 is made of carefully selected material with high strength and corrosion resistance, ensuring stable performance during long-term use. Loosening or rust will not affect the connection effect of the spring pressure plate 208, further guaranteeing the normal operation of the confocal dish adapter.

[0052] Specifically, such as Figures 9 to 12 As shown, the first end of the spring pressure plate 208 is connected to the second mounting groove 2052 by a fixing screw 207. A threaded hole 2054 is provided near the second end of the second mounting groove 2052, and the fixing screw 207 is screwed into the threaded hole 2054. The first end of the spring pressure plate 208 forms a U-shaped end, and the fixing screw 207 is connected to the U-shaped end. An internal threaded section is formed at the top of the inner wall of the confocal dish adapter base 201, and an external threaded section 2051 is formed at the bottom of the outer wall of the cover 205. The external threaded section 2051 and the internal threaded section are screwed together. A limiting groove 2042 is formed on the side of the limiting ring 204, and a first step is formed on the inner side wall of the confocal dish adapter base 201. The limiting groove 2042 is placed on the first step.

[0053] Preferably, the bottom of the cover 205 is provided with a pressure groove 2056, the bottom end of the second through hole 2055 is located in the pressure groove 2056, and the first through hole 2041 corresponds to the pressure groove 2056.

[0054] Better, such as Figures 9 to 12As shown, the inner wall of the confocal dish adapter base 201 has an internal groove thread 2011, and the inner wall of the confocal dish adapter base 201 has a third step 2012 and a fourth step 2013. The bottom of the confocal dish 202 is placed on the fourth step 2013, and the top edge of the confocal dish 202 rests on the third step 2012. An observation through hole 2014 is provided at the bottom of the confocal dish adapter base 201 to facilitate observation of the internal condition of the confocal dish 202.

[0055] Example 3, as Figures 13 to 14 As shown, the adapter is a tissue slide adapter, including: a tissue slide adapter base 302, the tissue slide adapter base 302 having a limiting hole 3021, the limiting hole 3021 having a sample carrier 301 placed therein, the tissue slide adapter base 302 having an observation hole 3022 at the bottom for easy observation of the sample; the sample carrier 301 being covered with a limiting washer 304, and the limiting washer 304 being fitted with a second sealing ring 303; The cover 205 is placed on the tissue section adapter base 302. The cover 205 of the multi-well plate adapter has the same structure as the cover of the confocal dish adapter in Embodiment 2, and will not be described again here. Two capillaries 206 are L-shaped and include a first section and a second section. The first section passes through the second through hole 2055 and its bottom end abuts against the sample carrier 301. The second section is placed in the first mounting groove 2053. The capillaries 206 are connected to the channel switching valve a and the channel switching valve b through pipes. One capillary 206 can be used as an injection port and the other capillary 206 can be used as a waste suction port. A pressure limiting component is installed on the top of the cover 205. The pressure limiting component has the same structure as the pressure limiting component of the confocal dish adapter in Embodiment 2, and will not be described again here.

[0056] Specifically, such as Figures 13 to 14 As shown, a limiting washer 304 is placed inside the tissue slice adapter base 302. The limiting washer 304 has a third through hole 3041. The limiting washer 304 and the sample carrier 301 are stacked to form a sample groove in the limiting hole 3021. The sample groove is used to place a sample. The second through hole 2055 of the cover 205 communicates with the sample groove. The limiting washer 304 is equipped with a second sealing ring 303. The second sealing ring 303 is located between the end of the cover 205 and the sample carrier 301, and the second sealing ring 303 is arranged around the third through hole 3041.

[0057] Preferably, the top of the inner wall of the limiting hole 3021 of the tissue slide adapter base 302 has an internal thread section 3023, and the bottom of the outer wall of the cover 205 has an external thread section 2051. The external thread section 2051 and the internal thread section 3023 are screwed together to fix the cover 205 and the tissue slide adapter base 302. This connection method is simple to operate, reliable in structure, and can ensure the stability of the experiment. An observation hole 3022 is provided at the bottom of the tissue slide adapter base 302 to facilitate observation of the sample preparation process.

[0058] A method of using an automated high-throughput immunostaining sample preparation system, comprising the following steps, wherein the method is performed using the automated high-throughput immunostaining sample preparation system as described above: Multiple sample preparation solutions are loaded into the sample supply assembly. These solutions include washing solution, fixative solution, PBS solution, blocking solution, primary antibody solution, WB solution, and secondary antibody solution. Several channels of channel switching valve c4 correspond to one sample preparation solution. Each channel of channel switching valve a and channel switching valve b corresponds to an adapter. Sample preparation parameters are set in the host computer. The host computer starts the peristaltic pump a5 through the communication module. The host computer opens the corresponding channel positions of channel switching valve a and channel switching valve c4 through the communication module so that the peristaltic pump a5 transfers the sample preparation liquid corresponding to the sample supply component into the corresponding adapter. The host computer starts the peristaltic pump b9 through the communication module, and opens the corresponding channel position of the corresponding channel switching valve b through the communication module, so that the peristaltic pump b9 transfers the liquid in the corresponding adapter into the waste liquid bottle.

[0059] The workflow of the automated sample preparation system of this invention during formal operation is as follows: Prepare all reagent solutions and load them into centrifuge tubes of different volumes; select the number of samples to be prepared (range 1-11) on the host computer software and set various parameters for each batch, such as the injection volume of each reagent, the corresponding incubation time, and the waste volume. Typically, the single injection volume of fixative solution, WB solution, blocking solution, and PBS solution is 500 μL, and the primary and secondary antibody solutions are 200 μL; Internal hardware control process: The host computer controls channel switching valves c4 and a through communication module a, switching to the corresponding centrifuge tube containing the reagent and the corresponding confocal dish adapter; then the host computer sends a start rotation command to the peristaltic pump a5; after a waiting time (waiting time = input volume ÷ set flow rate), the host computer sends a stop rotation command to the peristaltic pump a5; after another 5-second waiting time, click to start sample preparation on the host computer. The extra 5-second waiting time is due to the dead volume between the connecting tubes, so the actual aspiration volume time needs to be extended.

[0060] Flowchart of the automated sample preparation system: Step 1: Initialize the host computer variable j=0, where j represents the count of the sample preparation process; Step 2: Extract waste liquid, with an actual extraction volume of 700 μL. j = j + 1. When j = 1, proceed to Step 4; when j = 4, 7, or 10, proceed to Step 5; when j = 13, proceed to Step 6; when j = 16, proceed to Step 7; when j = 21, 24, 32, or 35, proceed to Step 9; when j = 27, proceed to Step 10; when j = 38, proceed to Step 11. Step 3: Switch channel switching valve a to channel 12, inject cleaning solution, j=j+1, and proceed to step 2 after the set cleaning time.

[0061] Step 4: Channel switching valve a switches sequentially from channel 1 to channel 11, injects fixative solution, j=j+1, and waits for the set action time before proceeding to step 3; Step 5: Channel switching valve a switches sequentially from channel 1 to channel 11, injects PBS solution, j=j+1, and waits for the set waiting time before proceeding to step 3; Step 6: Channel switching valve a switches sequentially from channel 1 to channel 11, injects sealing solution, j=j+1, and waits for the set sealing time before proceeding to step 3; Step 7: Channel switching valve a switches sequentially from channel 1 to channel 11, injects primary antibody solution, j=j+1, and waits for the set primary antibody incubation time before proceeding to step 8; Step 8: Channel switching valve b switches sequentially from channel 1 to channel 11 to extract waste liquid. The actual extracted volume is 280μL, j=j+1, and then proceeds to step 9. Step 9: Switch the channel switching valve a sequentially from channel 1 to channel 11, inject WB solution, j=j+1, and wait for the set incubation time before returning to step 3; Step 10: Switch the channel switching valve a from channel 1 to channel 11, inject the secondary antibody solution, j=j+1, and wait for the set secondary antibody incubation time before proceeding to step 8; Step 11: Switch the channel switching valve a from channel 1 to channel 11 and inject PBS solution. The PBS solution is used to keep the sample immersed in PBS to prepare for the next sample testing experiment. Proceed to Step 12. Step 12: j = j - 38, complete all antibody staining and sample preparation processes, and reset j to zero.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An automated high-throughput immunostaining sample preparation system, characterized in that, Includes a host computer, several sets of adapters, peristaltic pump a, peristaltic pump b, several communication modules, channel switching valve a, channel switching valve b, and channel switching valve c; The host computer is connected to peristaltic pump a and peristaltic pump b. Peristaltic pump b is connected to a waste liquid bottle. Peristaltic pump a is connected to a sample supply assembly via a channel switching valve c. The sample supply assembly is loaded with sample preparation solution. Several sets of adapters are disposed between channel switching valve a and channel switching valve b. Channel switching valve a is connected to peristaltic pump a, channel switching valve b is connected to peristaltic pump b, and channel switching valve a is connected to the waste liquid bottle. The communication module has 5 groups, namely communication module a, communication module b, communication module c, communication module d, and communication module e. Communication module a is set between the host computer and peristaltic pump a, communication module b is set between the host computer and peristaltic pump b, communication module d is set between the host computer and channel switching valve a, communication module e is set between the host computer and channel switching valve b, and communication module c is set between the host computer and channel switching valve c. The host computer sends a command to the peristaltic pump a through the communication module a, controlling it to draw the sample preparation solution from the sample supply component and deliver the sample preparation solution to the corresponding adapter through the channel switching valve c. The host computer controls the peristaltic pump b through the communication module b, and the peristaltic pump b transfers the liquid in the corresponding adapter into the waste liquid bottle; During sample preparation, channel switching valves a and b are switched under the control of the host computer via communication modules d and e, so that the sample preparation solution can flow through each adapter according to the preset path to complete the immunostaining step.

2. The automated high-throughput immunostaining sample preparation system according to claim 1, characterized in that, The adapter is a perforated plate adapter, which includes: A perforated plate having multiple perforations and slots; The cover plate has two insertion holes corresponding to each of the slots, and the cover plate has a mounting groove. The cover plate is disposed on the perforated plate. The pagoda connector has a first hole and a second hole, which are concentrically arranged. The inner diameter of the first hole is larger than the inner diameter of the second hole. The pagoda connector has an external thread. The top of the pagoda connector is connected to the channel switching valve a or the channel switching valve b through a pipe. A steel needle, wherein the steel needle is hollow and straight, and the first end of the steel needle is at an angle and the second end is at a flat angle; A limiting component is installed on the cover plate and is used to fix the position of the pagoda connector.

3. The automated high-throughput immunostaining sample preparation system according to claim 2, characterized in that, The second end of the steel needle is inserted and connected to the first hole of the pagoda connector; the limiting component includes a spring and a nut, the spring is sleeved on the pagoda connector, the spring is disposed between the pagoda connector and the cover plate, and the axial elastic force generated by the spring enables the first end of the steel needle to contact the bottom of the hole groove of the perforated plate.

4. The automated high-throughput immunostaining sample preparation system according to claim 3, characterized in that, The cover plate has two insertion holes for each of the slots. The pagoda connector passes through the insertion holes of the cover plate and is externally threaded to the nut so that the pagoda connector is fixed to the insertion holes of the cover plate. The top of one of the pagoda connectors passing through the insertion hole is connected to the channel switching valve a through a pipe, and the top of the other pagoda connector is connected to the channel switching valve b through a pipe.

5. The automated high-throughput immunostaining sample preparation system according to claim 1, characterized in that, The adapter is a confocal dish adapter, which includes: A confocal dish adapter base is provided, in which a confocal dish is placed. The confocal dish is covered with a limiting ring, and a first through hole is formed in the middle of the limiting ring. A boss is formed at the end of the limiting ring corresponding to the position of the first through hole, and a first sealing ring is fitted on the boss. The cover is placed on the confocal dish adapter base. The top of the cover has two first mounting slots. The cover has a second through hole. The top of the second through hole is connected to the first mounting slot. The cover has two second mounting slots. The two second mounting slots and the two first mounting slots are arranged in a one-to-one correspondence. The first end of the second mounting slot is connected to the first mounting slot. Two capillaries, each L-shaped, each including a first section and a second section, the first section passing through the second through hole, the bottom end of the first section abutting against the confocal dish, the second section placed in the first mounting groove, one of the capillaries being connected to the channel switching valve a via a pipe, and the other capillary being connected to the channel switching valve b via a pipe; A pressure limiting assembly is installed on the top of the cover body to limit the position of the second pipe segment. The pressure limiting assembly includes a spring plate, the first end of which is connected to the bottom of the second mounting groove and located near the second end of the second mounting groove, and the second end of which abuts against the cover body and covers the first mounting groove.

6. The automated high-throughput immunostaining sample preparation system according to claim 5, characterized in that, The first end of the spring pressure plate is connected to the second mounting groove by a fixing screw. The second mounting groove has a threaded hole near the second end. The fixing screw is screwed into the threaded hole. The first end of the spring pressure plate forms a U-shaped end, and the fixing screw is connected to the U-shaped end. An internal thread section is formed at the top of the inner wall of the confocal dish adapter base, and an external thread section is formed at the bottom of the outer wall of the cover. The external thread section and the internal thread section are screwed together. The side of the limiting ring forms a limiting groove, and the inner sidewall of the confocal dish adapter base forms a first step, with the limiting groove placed on the first step.

7. The automated high-throughput immunostaining sample preparation system according to claim 1, characterized in that, The adapter is a tissue slide adapter, which includes: A tissue slide adapter base has a limiting hole for placing a sample carrier. An observation hole is provided at the bottom of the tissue slide adapter base. A limiting gasket is provided on the sample carrier, and a second sealing ring is fitted on the limiting gasket. A cover body is provided on the tissue slide adapter base. The top of the cover body has two first mounting slots. The cover body has a second through hole. The top end of the second through hole is connected to the first mounting slot. The cover body has two second mounting slots. The two second mounting slots and the two first mounting slots are arranged in a one-to-one correspondence. The first end of the second mounting slot is connected to the first mounting slot. Two capillaries, each L-shaped, each including a first section and a second section, the first section passing through the second through hole, the bottom end of the first section abutting against the sample carrier, the second section placed in the first mounting groove, one of the capillaries being connected to the channel switching valve a via a pipe, and the other capillary being connected to the channel switching valve b via a pipe; A pressure limiting assembly is installed on the top of the cover body to limit the position of the second pipe segment. The pressure limiting assembly includes a spring plate, the first end of which is connected to the bottom of the second mounting groove and located near the second end of the second mounting groove, and the second end of which abuts against the cover body and covers the first mounting groove.

8. The automated high-throughput immunostaining sample preparation system according to claim 7, characterized in that, The first end of the spring pressure plate is connected to the second mounting groove by a fixing screw. The second mounting groove has a threaded hole near the second end. The fixing screw is screwed into the threaded hole. The first end of the spring pressure plate forms a U-shaped end, and the fixing screw is connected to the U-shaped end. An internal thread section is formed at the top of the inner wall of the limiting hole, and an external thread section is formed at the bottom of the outer wall of the cover. The external thread section and the internal thread section are screwed together.

9. A method of using an automated high-throughput immunostaining sample preparation system, characterized in that, Performing the method of use using an automated high-throughput immunostaining sample preparation system as described in any one of claims 1 to 8 includes the following steps: Multiple sample preparation solutions are loaded into the sample supply assembly. These solutions include washing solution, fixative solution, PBS solution, blocking solution, primary antibody solution, WB solution, and secondary antibody solution. Several channels of channel switching valve c correspond to one sample preparation solution. Each channel of channel switching valve a and channel switching valve b corresponds to an adapter. Sample preparation parameters are set in the host computer. The host computer starts the peristaltic pump a through the communication module. The host computer opens the corresponding channel positions of channel switching valve a and channel switching valve c through the communication module so that the peristaltic pump a transfers the sample preparation liquid corresponding to the sample supply component into the corresponding adapter. The host computer starts the peristaltic pump b through the communication module, and opens the corresponding channel position of the corresponding channel switching valve b through the communication module, so that the peristaltic pump b transfers the liquid in the corresponding adapter into the waste liquid bottle.

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