Automatic screening system for nucleic acid aptamers

Abstract

The invention discloses an automatic nucleic acid aptamer screening system which comprises a consumable stack, a first conveying track, a liquid treatment workstation, a second conveying track, a first grabbing structure, a sample treatment analysis module and a controller, the consumable stack is used for storing reagents and consumables, the liquid treatment workstation is used for treating liquid samples, and the second conveying track is used for conveying the liquid samples to the second conveying track. The first conveying track is used for conveying reagents or consumables stored in the consumable stack to the liquid treatment work station, the first grabbing structure is used for transferring liquid samples, and the second conveying track is used for conveying the liquid samples treated by the liquid treatment work station to the grabbing range of the first grabbing structure; the controller is electrically connected with the consumable stack, the first conveying rail, the liquid treatment workstation, the second conveying rail, the first grabbing structure and the sample treatment analysis module, the system can simultaneously treat the target number corresponding to the hole number according to the hole number of the perforated plate in the liquid treatment workstation, and the screening efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of nucleic acid aptamer screening, and in particular to an automated nucleic acid aptamer screening system. Background Art

[0002] Nucleic acid aptamers are a type of short single-stranded RNA or DNA oligonucleotide sequences that can form a partially complementary secondary structure, and further fold and entangle to form a more complex three-dimensional conformation on this basis. They can recognize specific target molecules, similar to antibodies, and are important functional molecules. At the same time, they have the special properties of nucleic acids such as amplification, sequencing and encoding, and have broad application prospects.

[0003] All aptamers are selected by in vitro evolution, which is called systematic evolution of ligands by exponential enrichment (SELEX). Classic SELEX usually includes incubation, separation and amplification. First, the random library is naturally folded into a variety of secondary structures, such as hairpins, pseudoknots, protrusions, G-quadruplexes, etc., and then incubated and bound to the target. Then, the unbound nucleic acid sequences are separated by different separation techniques to obtain the sequences that bind to the target. Subsequently, the sequences that bind to the target are eluted and amplified by PCR. Finally, the candidate sequences undergo multiple rounds of iterative screening, and they eventually dominate the library and are gradually enriched, and then the screened sequences are sequenced and analyzed.

[0004] Traditional SELEX technology usually relies on manual work and requires multiple rounds of screening to isolate aptamers with sufficient specificity and binding affinity, which has the disadvantages of low throughput, long time consumption, high operational error rate, poor stability, and low success rate, which greatly hinders the rapid development of this field.

[0005] The wide application of nucleic acid aptamers has promoted the continuous improvement of SELEX technology. In order to improve the screening efficiency and success rate, semi-automatic screening has gradually replaced traditional manual screening, such as semi-automatic screening instruments based on magnetic rods; the instrument has a matching deep-well plate and a magnetic rod sleeve, which is loaded on the mobile device of the semi-automatic instrument, and the magnetic rod sleeve can be extended into the deep-well plate to achieve mixing and magnetic suction operations. Semi-automatic screening reduces some manual experimental steps, such as incubation and cleaning, and can simultaneously complete the screening of more than a dozen targets, and the screening efficiency is increased by more than ten times. At the same time, the factors of human interference are also reduced to a minimum, which is very time-saving and labor-saving. However, semi-automatic screening still cannot free your hands, and some experimental operations still need to be performed manually, such as automated heating and elution, PCR amplification and single-stranded preparation; especially the quality inspection parts such as qPCR detection in screening process monitoring, fragment size analysis of single-stranded products, and concentration determination of single-stranded product pairs, which still cannot be automated. Moreover, semi-automatic screening can only realize the automation of some experimental links, and the entire screening experimental link cannot be realized in automatic and continuous operation, which consumes a lot of manpower and time. In addition, the semi-automated screening throughput is still low, and only a dozen targets can be screened in parallel, which greatly affects the screening process and efficiency. Therefore, people are in urgent need of new SELEX methods that are more efficient and faster. Summary of the invention

[0006] In order to overcome at least one defect of the above-mentioned prior art, the present invention provides an automated screening system for nucleic acid aptamers, which can solve the problem that the SELEX technology in the prior art needs to rely on manual implementation, has the disadvantages of low throughput, long time consumption, high manpower consumption, high operation error rate, poor stability, low success rate, etc.

[0007] The technical solution adopted by the present invention to solve the problem is:

[0008] An automated nucleic acid aptamer screening system, comprising:

[0009] Consumables stack, which is used to store reagents and consumables;

[0010] First transport track;

[0011] A liquid handling workstation, the liquid handling workstation is used for pipetting, mixing, dispensing and recovering liquid samples, and the first transport track is used for transporting reagents or consumables stored in the consumables stack to the liquid handling workstation;

[0012] Second transport track;

[0013] a first gripping structure, the first gripping structure is used to transfer the liquid sample, and the second transport track is used to transfer the liquid sample processed by the liquid processing workstation to the gripping range of the first gripping structure;

[0014] A sample processing and analysis module, which is used for processing liquid sample containers and analyzing liquid samples;

[0015] A controller, the controller being electrically connected to the consumables stack, the first transport track, the liquid handling workstation, the second transport track, the first gripping structure and the sample processing and analysis module;

[0016] Wherein, the sample processing and analysis module and the second transport track are both located within the gripping range of the first gripping structure.

[0017] By adopting the above scheme, the system can simultaneously process the number of targets corresponding to the number of wells in the multi-well plate in the liquid handling workstation, greatly improving the screening efficiency. Traditional single-target screening takes 2-3 weeks, while this system can complete the screening of the number of targets corresponding to the number of wells within 5-7 days. The entire system is controlled by a control program and runs quickly and accurately according to the set program, which minimizes human intervention and greatly improves the reproducibility of experimental results and experimental efficiency. The use of an automated screening system can expand the throughput of experimental design, avoid human errors, accelerate research progress, speed up the acquisition of research results, and help shorten the research and development cycle of new nucleic acid aptamers.

[0018] Further, the liquid handling workstation comprises:

[0019] First pipette;

[0020] A first cleaning station, the first cleaning station is used for cleaning the first pipette;

[0021] A vortex oscillation module, which is used for oscillating and mixing liquid samples;

[0022] PCR module, the PCR module is used to complete the PCR reaction;

[0023] Second pipette;

[0024] A second cleaning station, the second cleaning station is used for cleaning the second pipette;

[0025] A garbage station, the garbage station is used to collect waste generated by the first pipette and / or the second pipette;

[0026] Constant temperature incubator, which is used to incubate samples;

[0027] Among them, the first pipette, the first cleaning station, the vortex oscillation module, the PCR module, the second pipette, the second cleaning station, the garbage station and the constant temperature incubator are all electrically connected to the controller.

[0028] By adopting the above scheme, by integrating the first cleaning station, vortex oscillation module, PCR module, second cleaning station, garbage station and constant temperature incubator, and these modules are electrically connected to the controller, the nucleic acid aptamer automated screening system realizes the full process automation control from preliminary cleaning to final incubation. This design ensures the efficient cleaning, sufficient mixing, accurate PCR, re-cleaning to remove impurities, effective management of waste and stable incubation of samples at constant temperature during the screening process of liquid samples, thereby not only improving the screening efficiency, but also ensuring the consistency and reliability of the experimental results, reducing the errors introduced by human operation, enhancing the ability to accurately control the experimental process, and ultimately improving the overall quality and success rate of nucleic acid aptamer screening.

[0029] Furthermore, the liquid handling workstation also includes a second gripping structure for pipetting and transporting consumables.

[0030] By adopting the above scheme, the structure is used for pipetting and handling consumables, allowing the workstation to handle diverse experimental needs more flexibly. By adding a second gripping structure, the system can achieve more efficient automated operations, further reducing the chance of manual intervention and improving the stability and accuracy of the experimental process. In addition, this design allows the workstation to handle more experimental steps, enhancing the system's versatility and ability to handle complex experiments, thereby improving overall experimental efficiency while ensuring the consistency and reliability of experimental results.

[0031] Further, the first pipette is a 96-channel pipette, the first washing station is a 96-channel washing station, the second pipette is an 8-channel pipette, and the second washing station is an 8-channel washing station.

[0032] By adopting the above scheme, the first pipette is a 96-channel pipette, and the first cleaning station is a 96-channel washing station, which corresponds to the more common 96-well plate on the market; the second pipette is an 8-channel pipette, and the second cleaning station is an 8-channel washing station. The above structure enables the system to efficiently clean the tip of the first pipette and the tip of the second pipette, ensuring the accuracy and consistency of subsequent sample processing, thereby improving the reliability and quality of the screening results.

[0033] Furthermore, the sample processing and analysis module includes:

[0034] A film sealing machine, which is used to seal the container of the liquid sample;

[0035] Film peeling machine, which is used to remove the sealing film on the container of liquid sample;

[0036] Centrifuge, which is used to process liquid samples;

[0037] Sample analysis module: the sample analysis module is used for counterfeit analysis of intermediate products or final products in the screening process;

[0038] Among them, the film sealing machine, film tearing machine, centrifuge and sample analysis module are all electrically connected to the controller.

[0039] By adopting the above solution, the automatic sealing and tearing of the liquid sample container can be realized to ensure the sealing and pollution prevention of the sample during the processing; the centrifuge is responsible for the efficient centrifugal processing of the sample to ensure the separation and concentration of the sample; the sample analysis module is used to accurately analyze the intermediate products or final products in the screening process, thereby ensuring the automation and efficiency of the entire screening process and the accuracy and reliability of the results. Through this highly integrated design, not only the throughput of the experiment is improved, but also the errors caused by human operation are reduced, and the consistency of the experimental results and the controllability of the experimental process are enhanced.

[0040] Furthermore, the sample analysis module includes:

[0041] A fragment analyzer, wherein the fragment analyzer (641) is used for analyzing nucleic acid fragments during the nucleic acid aptamer screening process;

[0042] Fluorescence quantitative PCR instrument, which is used to perform quantitative analysis of nucleic acids in liquid samples;

[0043] Microplate reader, which is used to perform quantitative and / or qualitative analysis on liquid samples;

[0044] Among them, the fragment analyzer, the fluorescence quantitative PCR instrument and the enzyme marker are all electrically connected to the controller.

[0045] By adopting the above scheme, the system can achieve efficient screening of nucleic acid aptamers, accurate quantitative analysis of nucleic acids, and quantitative and qualitative analysis of samples. The fragment analyzer can perform high-resolution fragment analysis of nucleic acid aptamers, the fluorescent quantitative PCR instrument can monitor and quantify the concentration of nucleic acids in real time, and the microplate reader can perform multifunctional quantitative and qualitative analysis of samples. This highly integrated sample analysis module not only improves the automation of screening and analysis, but also ensures the accuracy and reliability of experimental results, reduces human operation errors, and thus significantly improves experimental efficiency and data consistency.

[0046] Furthermore, the microplate reader includes a light absorption module, a chemiluminescence module and a fluorescence intensity module. The wavelength range of the light absorption module is 230-1000nm, the wavelength range of the chemiluminescence module is 300-800nm, and the wavelength range of the fluorescence intensity module is 250-850nm.

[0047] By adopting the above scheme, the wavelength range of the light absorption module is 230-1000nm, which can be used to detect different types of samples, such as protein, DNA / RNA, etc., providing a wide range of light absorption detection capabilities;

[0048] The wavelength range of the chemiluminescence module is 300-800nm, which is suitable for chemiluminescence detection. It can be used to detect enzyme activity, immune response, etc., providing a highly sensitive detection method;

[0049] The fluorescence intensity module has a wavelength range of 250-850nm and is suitable for the detection of fluorescently labeled samples, such as fluorescent proteins, nucleic acids labeled with fluorescent dyes, etc., providing high specificity and sensitivity of fluorescence detection capabilities.

[0050] Furthermore, the first grasping structure is a four-axis robotic arm.

[0051] By adopting the above solution, it is possible to efficiently transport consumables between devices at different heights. The four-axis robotic arm can not only complete complex grasping movements, but also ensure accurate positioning and placement between workstation peripherals. This design greatly improves the system's operating efficiency and automation level when handling diverse experimental tasks, reduces the possibility of human intervention, and thus ensures the consistency of the experimental process and the reliability of the results.

[0052] Furthermore, a plurality of shelves and a rotating mechanism for driving the plurality of shelves to rotate are provided in the consumables stack. The rotating mechanism can drive the plurality of shelves to rotate, and the rotating mechanism is electrically connected to the controller.

[0053] By adopting the above solution, the consumables stack can automatically and intelligently store and retrieve consumables, and the controller can accurately control the rotating mechanism to realize automatic scheduling of consumables, thereby improving the efficiency and accuracy of consumables management. This design not only reduces the frequency of manual operation and the risk of operational errors, but also makes the operation of the consumables stack more convenient and efficient, thereby improving the automation level of the entire system and the fluency of the experimental process.

[0054] In summary, the present invention provides an automated nucleic acid aptamer screening system with the following technical effects:

[0055] 1. Through the combination of consumables stack, first transport track, liquid handling workstation, second transport track, first grabbing structure, sample processing and analysis module and controller, a highly automated system is formed. This integrated design enables the entire screening process from consumables preparation to final sample analysis to be completed without human intervention, greatly improving experimental efficiency;

[0056] 2. The four-axis robotic arm is used as the first grasping structure, which can realize the transportation of consumables between devices at different heights outside the workstation. This high-precision robotic arm is connected to the controller and can accurately perform tasks according to the preset program, greatly reducing the need for manual intervention and improving the automation level of the experimental process;

[0057] 3. The sample analysis module includes a fragment analyzer, a fluorescence quantitative PCR instrument, and an enzyme-labeled instrument, all of which are electrically connected to the controller. This integrated design enables the system to complete multiple analysis tasks on one platform, from nucleic acid fragment analysis to quantitative PCR to enzyme-labeled instrument detection, covering the key steps in the screening process and ensuring data consistency and experimental coherence;

[0058] 4. The consumables stack is equipped with multiple shelves and a rotating mechanism that drives these shelves to rotate. The storage and access of consumables can be accurately controlled by the controller. This design not only improves the efficiency of consumables management, but also makes the scheduling of consumables more intelligent and reduces errors caused by improper human operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a structural schematic diagram of the present invention;

[0060] Figure 2 This is a schematic diagram of the setting status of the first manipulator and the second manipulator in the liquid processing workstation of the present invention.

[0061] Among them, the meanings of the figure marks are as follows: 1. Consumables stack; 2. First transport track; 3. Liquid handling workstation; 31. First cleaning station; 32. Vortex oscillation module; 33. PCR module; 34. Second cleaning station; 35. Garbage station; 36. Constant temperature incubator; 37. Second grabbing structure; 371. First manipulator; 372. Second manipulator; 38. First pipette; 39. Second pipette; 4. Second transport track; 5. First grabbing structure; 61. Film sealing machine; 62. Film tearing machine; 63. Centrifuge; 641. Fragment analyzer; 642. Fluorescence quantitative PCR instrument; 643. Microplate reader. DETAILED DESCRIPTION

[0062] For better understanding and implementation, the technical solutions in the embodiments of the present invention will be clearly and completely described and discussed below in conjunction with the accompanying drawings of the present invention. Obviously, what is described here is only a part of the examples of the present invention, not all the examples. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0063] In order to facilitate the understanding of the embodiments of the present invention, the following will be further explained by taking specific embodiments as examples in conjunction with the drawings, and each embodiment does not constitute a limitation on the embodiments of the present invention.

[0064] In the description of the present invention, it should be noted that the terms "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, they cannot be understood as limiting the present invention.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0066] See also Figure 1-Figure 2 The present invention discloses an automated screening system for nucleic acid aptamers, comprising a consumables stack 1, a first transport track 2, a liquid processing workstation 3, a second transport track 4, a first gripping structure 5, a sample processing and analysis module and a controller. The consumables stack 1 is used to store reagents and consumables. The liquid processing workstation 3 is used for pipetting, mixing, distributing and recovering liquid samples, and the first transport track 2 is used to transfer the reagents or consumables stored in the consumables stack 1 to the liquid processing workstation 3. The first gripping structure 5 is used to transfer liquid samples, and the second transport track 4 is used to transfer the liquid samples processed by the liquid processing workstation 3 to the gripping range of the first gripping structure 5. The sample processing and analysis module is used for processing liquid sample containers and analyzing liquid samples. The controller is electrically connected to the consumables stack 1, the first transport track 2, the liquid processing workstation 3, the second transport track 4, the first gripping structure 5 and the sample processing and analysis module respectively. The sample processing and analysis module and the second transport track 4 are both located within the gripping range of the first gripping structure 5.

[0067] Further, the liquid handling workstation 3 includes a first pipette, a first cleaning station 31, a vortex oscillation module 32, a PCR module 33, a second pipette, a second cleaning station 34, a garbage station 35 and a constant temperature incubator 36, wherein the first cleaning station 31 is used for preliminary cleaning of the liquid sample. The vortex oscillation module 32 is used for oscillating and mixing the liquid sample. The PCR module 33 is used to complete the PCR reaction. The second cleaning station 34 is used for secondary cleaning of the liquid sample. The garbage station 35 is used to collect and process waste generated during the screening process. The constant temperature incubator 36 is used to incubate the sample. Among them, the first pipette, the first cleaning station 31, the vortex oscillation module 32, the PCR module 33, the second pipette, the second cleaning station 34, the garbage station 35 and the constant temperature incubator 36 are all electrically connected to the controller.

[0068] Specifically, by integrating the first pipette, the first cleaning station 31, the vortex oscillation module 32, the PCR module 33, the second pipette, the second cleaning station 34, the garbage station 35 and the constant temperature incubator 36, and these modules are electrically connected to the controller, the nucleic acid aptamer automated screening system realizes the full process automation control from preliminary cleaning to final incubation. This design ensures the efficient cleaning, sufficient mixing, accurate PCR, re-cleaning to remove impurities, effective management of waste and stable incubation of samples at a constant temperature during the screening process of liquid samples, thereby not only improving the screening efficiency, but also ensuring the consistency and reliability of the experimental results, reducing the errors introduced by human operation, enhancing the ability to accurately control the experimental process, and ultimately improving the overall quality and success rate of nucleic acid aptamer screening.

[0069] Furthermore, the liquid handling workstation 3 further includes a second gripping structure 37 for transferring liquid and transporting consumables. The specific equipment modules included in the corresponding liquid handling workstation 3 should all be arranged within the gripping range of the second gripping structure 37 .

[0070] Specifically, the structure is used for pipetting and handling consumables, so that the workstation can handle diverse experimental needs more flexibly. By adding the second gripping structure 37, the system can achieve more efficient automated operation, further reducing the chance of manual intervention and improving the stability and accuracy of the experimental process. In addition, this design allows the workstation to handle more experimental steps, enhancing the system's versatility and ability to handle complex experiments, thereby improving the overall experimental efficiency while ensuring the consistency and reliability of the experimental results.

[0071] In some embodiments, the first pipette is a 96-channel pipette, the first washing station 31 is a 96-channel washing station, the second pipette is an 8-channel pipette, and the second washing station 34 is an 8-channel washing station.

[0072] Specifically, the first pipette is a 96-channel pipette, and the first cleaning station 31 is a 96-channel washing station, which corresponds to the more common 96-well plate on the market. The second pipette is an 8-channel pipette, and the second cleaning station 34 is an 8-channel washing station. The above structure enables the system to efficiently clean the tips of the first pipette and the second pipette, thereby ensuring the accuracy and consistency of subsequent sample processing, thereby improving the reliability and quality of the screening results.

[0073] In some embodiments, when the first pipette or the second pipette transfers liquid, salt crystals may exist in the gun tip of the first pipette or the second pipette. For the above situation, three single-hole reagent reservoirs corresponding to the first pipette can be set at the first pipette, and the three single-hole reagent reservoirs are sequentially added with water, 75% alcohol and water, and placed within the range where the first pipette can move, and then the first pipette is moved and soaked in water, 75% alcohol and water for two hours respectively, to complete the dissolution of the salt crystals, thereby improving the accuracy of the volume of each channel imbibition in the first pipette. Similarly, within the range of movement of the second pipette, three corresponding single-hole reagent reservoirs are set, and the three single-hole reagent reservoirs are sequentially added with water, 75% alcohol and water, and placed within the range where the second pipette can move, and then the second pipette is moved and soaked in water, 75% alcohol and water for two hours respectively, to complete the dissolution of the salt crystals, thereby improving the accuracy of the volume of each channel imbibition in the second pipette.

[0074] In addition, in the present embodiment, the second gripping structure 37 includes a first manipulator 371 and a second manipulator 372, wherein the first pipette is mounted on the first manipulator 371, and the second pipette is mounted on the second manipulator 372, so that the first pipette is driven by the driving component of the first manipulator 371 without affecting the normal gripping process of the first manipulator 371, thereby saving part of the driving component. The working principle and beneficial effect of the second pipette being mounted on the second manipulator 372 are the same as the working principle and beneficial effect of the first pipette being mounted on the first manipulator 371, and will not be described in detail here.

[0075] In some embodiments, when the first pipette or the second pipette is transferring liquid, the aspirated volume of some channels may be reduced in accuracy due to bubbles. To address the above problem, the liquid storage barrels connected to the first pipette and the second pipette may be filled with independent pure water and 75% alcohol, respectively. When bubbles are generated in the channel, the first pipette or the second pipette that generates the bubbles is controlled by a solenoid valve or other flow guide device to replace the extracted liquid from pure water with 75% alcohol. After cleaning for 3-7 minutes, the channel is detected manually or by a visual recognition device to see if there are still bubbles. After cleaning is completed, the liquid extracted by the first pipette or the second pipette is changed to pure water for subsequent experimental operations.

[0076] In some embodiments, the sample processing and analysis module includes a film sealing machine 61, a film tearing machine 62, a centrifuge 63 and a sample analysis module, the film sealing machine 61 is used to seal the container of the liquid sample, the film tearing machine 62 is used to remove the sealing film on the container of the liquid sample, the centrifuge 63 is used to process the liquid sample, and the sample analysis module is used to analyze the intermediate products or final products in the screening process, wherein the film sealing machine 61, the film tearing machine 62, the centrifuge 63 and the sample analysis module are all electrically connected to the controller.

[0077] Specifically, it can realize automatic film sealing and film tearing of liquid sample containers to ensure the sealing and pollution prevention of samples during the processing; the centrifuge 63 is responsible for efficient centrifugal processing of samples to ensure the separation and concentration of samples; the sample analysis module is used to accurately analyze the intermediate products or final products in the screening process, thereby ensuring the automation and efficiency of the entire screening process and the accuracy and reliability of the results. Through this highly integrated design, not only the throughput of the experiment is improved, but also the errors caused by human operation are reduced, and the consistency of the experimental results and the controllability of the experimental process are enhanced.

[0078] In addition, the centrifuge 63 can realize workstation integration and can centrifuge PCR deep well plates, shallow well plates and other plates. The rotor of the centrifuge 63 can centrifuge at least 2 microplates and provide a centrifugal force of 3000RPM / 1000xg. The centrifuge 63 can be used online or offline. According to the preset screening method program, the reagents and consumables that need to be centrifuged are centrifuged here.

[0079] The liquid handling workstation 3 is also equipped with a PCR instrument, which can realize the second gripping structure 37 to carry the PCR plate between the PCR instrument and the workstation table. The automation system can control the opening and closing of the equipment and run the PCR temperature control program. The plate is sealed with a heatable plate cover without sealing film. It is compatible with full skirt and half skirt PCR plates, and the temperature control range is 4-105 degrees. In the PCR amplification stage of the screening process, automated operation can be realized.

[0080] Further, the sample analysis module includes a fragment analyzer 641, a fluorescence quantitative PCR instrument 642 and an enzyme marker 643, wherein the fragment analyzer 641 is used for screening nucleic acid aptamers. The fluorescence quantitative PCR instrument 642 is used for quantitative analysis of nucleic acids in liquid samples. The enzyme marker 643 is used for quantitative and / or qualitative analysis of liquid samples. Among them, the fragment analyzer 641, the fluorescence quantitative PCR instrument 642 and the enzyme marker 643 are all electrically connected to the controller. And the fragment analyzer 641 and the enzyme marker 643 can realize operations such as band size and concentration detection of the final product ssDNA of a round of screening.

[0081] In this embodiment, the fragment analyzer 641 uses automated parallel capillary electrophoresis technology, and can analyze DNA and RNA samples around the clock through an automated system. The system can use quantitative kits to analyze NGS libraries, large fragment diffuse bands, genomic DNA, RNA, and small RNA. Qualitative kits can also be used to analyze mutations or enzymatic DNA produced by PCR amplicons, SSR / microsatellites, and CRISPR. More than 2,400 samples can be analyzed every 24 hours, and a wide range of DNA and RNA fragments can be distinguished. The resolution of fragments below 300bp is as low as 3bp. In the screening process, the obtained ssDNA product needs to ensure the quality of the band size. The system integration of this instrument can automatically realize the electrophoresis experiment of the single-stranded band size.

[0082] Fluorescence quantitative PCR instrument 642 needs to be integrated with the workstation. It needs to have: 96-well plate reaction volume: 20-100ul, maximum heating rate: 4.8℃ / s, maximum cooling rate: 2.5℃ / s, excitation light source: standard xenon lamp, detection device: cold CCD. Filter: excitation wavelength (450nm, 483nm, 523nm, 558nm, 615nm) emission wavelength (500nm, 533nm, 568nm,

[0083] 610nm, 640nm, 670nm) Software functions: absolute quantification, Tm Calibration, relative quantification with efficiency correction, genotyping, gene scanning, product identification, etc. In the qPCR monitoring experiment of the screening process, qPCR quantitative analysis is required for the washing and elution products of the positive screen in order to monitor the enrichment of the screening library in real time.

[0084] The microplate reader 643 can be used for fully automatic online reading and automatic real-time calculation, thereby realizing online detection of nucleic acid concentration, and the concentration value can be directly used in subsequent experiments. The microplate reader 643 can be used online or offline as needed.

[0085] Specifically, the above structure enables the system to achieve efficient screening of nucleic acid aptamers, accurate quantitative analysis of nucleic acids, and quantitative and qualitative analysis of samples. The fragment analyzer 641 can perform high-resolution fragment analysis of nucleic acid aptamers, the fluorescent quantitative PCR instrument 642 can monitor and quantify the concentration of nucleic acids in real time, and the microplate reader 643 can perform multifunctional quantitative and qualitative analysis of samples. This highly integrated sample analysis module not only improves the degree of automation of screening and analysis, but also ensures the accuracy and reliability of experimental results, reduces human operation errors, and thus significantly improves experimental efficiency and data consistency.

[0086] In this embodiment, the microplate reader 643 includes a light absorption module, a chemiluminescence module and a fluorescence intensity module. The wavelength range of the light absorption module is 230-1000nm, the wavelength range of the chemiluminescence module is 300-800nm, and the wavelength range of the fluorescence intensity module is 250-850nm.

[0087] Specifically, the wavelength range of the light absorption module is 230-1000nm, which can be used to detect different types of samples, such as proteins, DNA / RNA, etc., providing a wide range of light absorption detection capabilities. The wavelength range of the chemiluminescence module is 300-800nm, which is suitable for chemiluminescence detection and can be used to detect enzyme activity, immune response, etc., providing a highly sensitive detection method. The wavelength range of the fluorescence intensity module is 250-850nm, which is suitable for the detection of fluorescently labeled samples, such as fluorescent proteins, nucleic acids labeled with fluorescent dyes, etc., providing highly specific and sensitive fluorescence detection capabilities.

[0088] In this embodiment, in order to provide more options for the consumables stack 1 and increase the degree of automation of the entire system, the first grasping structure 5 is a four-axis robotic arm, which can realize efficient handling of consumables between devices at different heights. The four-axis robotic arm can not only complete complex grasping actions, but also ensure accurate positioning and placement between workstation peripheral devices. Such a design greatly improves the operating efficiency and automation level of the system when handling diversified experimental tasks, reduces the possibility of human intervention, and thus ensures the consistency of the experimental process and the reliability of the results.

[0089] In some embodiments, a plurality of shelves and a rotating mechanism for driving the plurality of shelves to rotate are provided in the consumable stack 1 . The rotating mechanism can drive the plurality of shelves to rotate, and the rotating mechanism is electrically connected to the controller.

[0090] Specifically, the consumables stack 1 uses multiple shelves and a rotating mechanism to enable the consumables stack 1 to automatically and intelligently store and retrieve consumables. The controller precisely controls the rotating mechanism to achieve automatic scheduling of consumables, thereby improving the efficiency and accuracy of consumables management. This design not only reduces the frequency of manual operation and the risk of operational errors, but also makes the operation of the consumables stack 1 more convenient and efficient, thereby improving the automation level of the entire system and the fluency of the experimental process.

[0091] In this embodiment, each shelf can store 21 microplates (including covers) or 7 pipette tip boxes or 10 deep-well plates; it can rotate 360 ​​degrees, and the built-in spatula can automatically pick up the plates and lift them to the designated plate positions of the designated shelves. Before the screening experiment begins, the required pipette tips, deep-well plates, 384qPCR plates, 96PCR plates, libraries with good renaturation, screening pressure buffer, qPCR and PCR Mix and other reagents and consumables are placed in the stack according to the preset method procedures to prepare for the screening.

[0092] The working principle of the above structure is:

[0093] S1. Consumables preparation stage: Before the screening begins, place the prepared reagents and consumables (such as gun tips, deep-well plates, 384qPCR plates, 96PCR plates, denatured libraries, screening pressure buffer, qPCR and PCR Mix, etc.) in consumables stack 1 according to the written method program.

[0094] S2. Experiment start: After clicking Start, the automated workstation starts running according to the pre-programmed screening method program.

[0095] S3, reagent and consumables transportation: the first transport track 2 retrieves the reagents or consumables required for the experiment from the consumables stack 1 and transports them to the liquid handling workstation 3.

[0096] S4, operation of liquid handling workstation 3: After receiving the reagent consumables from the first transport track 2, the liquid handling workstation 3 starts to perform operations such as pipetting, mixing, dispensing, heating elution, magnetic removal of supernatant, PCR amplification, and single-stranded preparation. These operations include but are not limited to:

[0097] Oscillating and mixing: The liquid sample is oscillated and mixed by the vortex oscillation module 32 .

[0098] Temperature controlled heating: The PCR module 33 controls the temperature cycle of the PCR reaction.

[0099] Cleaning: The first cleaning station 31 and the second cleaning station 34 perform preliminary cleaning and secondary cleaning on the liquid sample respectively.

[0100] Magnetic separation: The magnetic stand module is used to separate magnetic beads.

[0101] Constant temperature incubation: The constant temperature incubator 36 is used to incubate the samples.

[0102] Waste disposal: Waste station 35 is used to collect and dispose of waste generated during the screening process.

[0103] S5, sample transfer: the processed liquid sample is transferred to the grasping range of the first grasping structure 5 (four-axis robot arm) through the second transport track 4.

[0104] S6, sample analysis: The first grabbing structure 5 transfers the processed liquid sample to the sample processing and analysis module for operations such as film sealing, film tearing, and centrifugation. The sample processing and analysis module includes:

[0105] Sealing machine 61: used for sealing the container of the liquid sample.

[0106] Film peeling machine 62: used for removing the sealing film on the liquid sample container.

[0107] Centrifuge 63: used to process liquid samples.

[0108] Sample analysis module: includes a fragment analyzer 641, a fluorescence quantitative PCR instrument 642 and an enzyme marker 643, which are used to analyze the intermediate products or final products in the screening process.

[0109] S7. Automated analysis: During the screening process, the system automatically performs analytical experiments according to the preset procedures, for example:

[0110] The washed eluted product after positive screening is automatically added to the qPCR Mix, mixed, and then automatically placed into the qPCR instrument by a robotic arm for qPCR operation.

[0111] After the single-stranded strands are prepared, part of the volume of each sample is automatically aspirated and added to the corresponding buffer. After mixing, the sample is automatically placed in the 5400 fragment analyzer 641 and the microplate reader 643 by a robotic arm for single-stranded strand size and concentration analysis experiments.

[0112] S8. Real-time monitoring and adjustment: These real-time monitoring data can not only be used to monitor the process status of SELEX, but also can be used to adjust the experimental parameters of each round of SELEX in a timely and accurate manner to greatly improve the success rate of target screening.

[0113] S9. Data recording and analysis: The system is equipped with data management software to record all process data and set filtering conditions to filter information. It supports real-time data call, realizes automatic call of sample number, volume, concentration, OD value read by enzyme label, etc., and automatically analyzes the data, and performs subsequent target sample selection or concentration homogenization experimental operations in real time.

[0114] Of course, the nucleic acid aptamer automated screening system provided by the present invention can also perform other experiments, the specific experiments are:

[0115] Automated library construction experiment (1-96 sample library construction)

[0116] PCR product purification specifically includes the following steps:

[0117] a. The amplified PCR product is purified by magnetic beads to recover bands above 70 bp. This method is suitable for purifying 1-96 samples. Of course, other adaptive adjustments can be made by adjusting the number of channels of the first pipette 38 and the first cleaning station 31.

[0118] b. Take a 2 cm high deep well plate matched with the liquid handling workstation 3, and place the magnetic beads equilibrated to room temperature. The best ratio of the amount of PCR product to the volume of magnetic beads is 1:1.8. Of course, different proportions of magnetic beads can also be added according to the size of the recovered product.

[0119] c. The first pipette transfers part or all of the PCR products from the 96-well PCR plate to the above-mentioned deep-well plate containing magnetic beads.

[0120] d. The deep well plate was transferred to an oscillator through the second grabbing structure 37, and mixed by shaking at 800 RPM for 1 min, and incubated at room temperature for 5 min to allow the DNA to bind to the magnetic beads.

[0121] e. The deep well plate is then transferred from the oscillator to the module containing the magnetic frame by the second grabbing structure 37, and allowed to stand for 3 minutes. After the solution is clarified, the first pipette removes the supernatant.

[0122] f. Keep the sample on the magnetic rack all the time. Use the first pipette to transfer 200ul from the reagent tank containing 80% ethanol to the deep-well plate. Let it stand for 30 seconds. After rinsing the magnetic beads, remove the supernatant.

[0123] g. Repeat step f once, rinsing twice in total, and absorb the remaining liquid.

[0124] h. Keep the sample on the magnetic rack and dry the magnetic beads at room temperature for about 5 minutes.

[0125] i. The deep well plate is transferred from the magnetic stand module to the common module by the second gripping structure 37, and an appropriate amount of water is added from the reagent tank containing sterile enzyme-free water by the first pipette.

[0126] j. Then transfer the deep-well plate from the ordinary module to the oscillator module through the second grabbing structure 37, vortex and oscillate at 1000RPM to mix thoroughly, let it stand at room temperature for 2 minutes, and then transfer it to the magnetic rack and let it stand for 3 minutes. After the solution is clarified, the first pipette uses a new 96-channel tip to carefully aspirate the supernatant into a new 2 cm deep-well plate to complete the purification of the PCR product.

[0127] The automated library construction experiment includes the following steps:

[0128] a. End repair: The first pipette sucks 100 ng of the purified PCR product and adds it to the PCR plate containing the end repair mixed reagent. After suction mixing, the PCR plate is transferred to the second transport track by the second grabbing structure 37. The surrounding external mechanical arms or the first grabbing structure 5 grab the PCR plate and place it in the film sealing machine for sealing. Then, it is transferred to the liquid handling workstation 3. The PCR plate is transferred to the PCR module by the first grabbing structure 5 at 20°C for 30 min; 65°C for 30 min; 4°C for ∞. The hot cover temperature is 75°C.

[0129] b. Connector connection: After the reaction is completed, the PCR plate is grabbed from the PCR module and transferred to the second transport track. The external mechanical arm or the first grabbing structure 5 grabs the PCR plate and places it in the film tearing machine to tear the film, and then it is transferred to the liquid handling workstation 3. The first pipette absorbs the mixed reagent for connector connection, adds it to the PCR plate, and mixes it by suction. The same operation is performed as above, and the film is sealed and transferred to the PCR module, 20℃, 15min; 4℃, ∞. No hot cover is provided.

[0130] c. Magnetic bead purification: After the reaction is completed, tear off the membrane of the PCR plate and transfer it to the liquid handling workstation 3 in the same way as above; purify the product after the above-mentioned connector connection in the same way as the PCR product purification operation.

[0131] d. Library amplification: First, use a pipette to add the mixed reagent for library amplification (containing Index connector) to the connector-connected and purified product in the previous step. After aspiration and mixing, place the mixture in a film sealing machine for sealing. Then, perform 6 cycles at 98°C for 10s, 60°C for 30s, and 72°C for 30s.

[0132] e. Second magnetic bead purification: Same as the magnetic bead purification step c above.

[0133] The nucleic acid aptamer automated screening system provided by the present invention can also perform magnetic bead method nucleic acid extraction and quantitative analysis experiments, specifically comprising the following steps:

[0134] a. Lysis: The samples were divided and placed in a 96-deep-well plate. A certain volume of lysis solution was aspirated from the reagent tank containing the lysis solution with a pipette and transferred to the sample deep-well plate. The deep-well plate was then transferred to an oscillator for thorough oscillation and mixing, and then transferred to a 4°C constant temperature incubation module for 30 min.

[0135] b. Magnetic bead binding: First, use a pipette to transfer a certain volume of magnetic beads from the reagent tank containing the magnetic beads and place it in the sample deep well plate. Vortex and mix thoroughly, and let it stand at room temperature for 5 minutes.

[0136] c. Washing: The deep-well plate is transferred to the module containing the magnetic frame by the second grabbing structure 37, and allowed to stand for 3 minutes. After the solution is clarified, the first pipette removes the supernatant. Keeping the sample on the magnetic frame, the first pipette draws 200u l from the reagent tank containing 80% ethanol and transfers it to the deep-well plate, and allows to stand for 30 seconds. After rinsing the magnetic beads, the supernatant is removed. Repeat the washing operation once. Then keep the sample on the magnetic frame and dry the magnetic beads at room temperature for about 5 minutes.

[0137] d. Elution: The deep-well plate is transferred from the magnetic stand module to the common module through the second grabbing structure 37, and the first pipette adds an appropriate amount of water from the reagent tank containing sterile enzyme-free water. The deep-well plate is then transferred from the common module to the oscillator module through the second grabbing structure 37, vortexed at 1000RPM to mix thoroughly, and allowed to stand at room temperature for 2 minutes. It is then transferred to the magnetic stand and allowed to stand for 3 minutes. After the solution is clarified, the first pipette uses a new 96-channel tip to absorb the supernatant into a new 2 cm deep-well plate.

[0138] e. Qualitative and quantitative analysis: A portion of the eluted nucleic acid product is added to the corresponding reagent of the fragment analyzer, and then the analyte is transferred to the 5400 fragment analyzer 641 through the external robotic arm or the first grabbing structure 5 to perform qualitative and quantitative analysis on the nucleic acid sample.

[0139] The nucleic acid aptamer automated screening system provided by the present invention can also automatically dilute samples, specifically comprising the following steps:

[0140] a. The second pipette of the liquid handling workstation 3 has 8 channels, each of which can pneumatically control different pipetting volumes, and can automatically dilute 96 samples of different concentrations to the same concentration.

[0141] b. First, place the first pipette, the sample PCR plate, the deep-well plate containing the diluent, and the empty deep-well plate for dilution in the common module of the liquid handling workstation 3 within the operating coverage of the second pipette.

[0142] c. Use Excel to determine the concentration and total volume of sample dilution, calculate the volume of each sample, and the dilution corresponding to each sample.

[0143] d. Then, the standardized calculated Excel table is imported into the method program written in the controller of the liquid handling workstation 3, and the amount of each sample and the amount of diluent to be taken are set, and added to the empty deep-well plate to complete the automated dilution of the sample.

[0144] The nucleic acid aptamer automated screening system provided by the present invention can also perform automated sample processing, specifically including the following types of operations:

[0145] a. Automated plate transfer: The liquid handling workstation 3 includes a first manipulator 371 and a 96-channel first pipette mounted on the first manipulator 371, a second manipulator 372 and an 8-channel second pipette mounted on the second manipulator 372. The first pipette and the second pipette can be operated simultaneously to improve efficiency. The first manipulator 371 and the second manipulator 372 are also equipped with grippers to achieve full table coverage inside the liquid handling workstation 3. The gripper can be used to carry consumables such as microplates and pipette tips. The gripper is designed with an oblique side clamp, which can adjust the gripper strength according to the type and weight of the consumables to ensure the safety of the gripper.

[0146] b. Automated sample loading and liquid transfer: Liquid handling workstation 3 is equipped with a 96-channel first pipette and an 8-channel second pipette, in which each channel of the 8-channel pipette of the second pipette can be independently controlled, and the spacing can be automatically adjusted according to the consumables with different hole spacings. All channels have a liquid level detection function. Each channel can be calibrated independently. The 8-channel pipette is liquid-driven, with a pipetting volume range of 1-1000u l and a pipetting accuracy of 1u l CV <= 7%. The 96-channel pipette of the first pipette is an air displacement pipette with a pipetting volume range of 1-300u l and a pipetting accuracy of 1u lCV <= 5%. The 96-channel pipette can realize 96-channel, 12-channel, 8-channel and single-channel loading, and can also realize loading with any number and layout of pipette tips between 1-96, which is convenient for 96 channels to realize tube plate transfer, serial dilution and other operations.

[0147] c. Automatic vortex oscillation: The liquid handling workstation 3 provides a vortex oscillation module 32, which has an automatic sensing lock with a maximum speed of ≥1800rpm, and can sense whether the multi-well plate is placed correctly; the program comes with a variety of oscillation modes, and the oscillation mode can also be adjusted according to the customer's experimental requirements to meet different needs in the experiment.

[0148] d. Automatic constant temperature incubation: The liquid handling workstation 3 is equipped with a constant temperature incubation module 36. The constant temperature incubation module 36 adopts the Peltier principle, with a temperature range of 4-100 degrees and a temperature accuracy of ±0.3°C. The temperature can be set either through the remote control module or through the controller of the liquid handling workstation 3.

[0149] e. Automated cleaning of gun tips: The liquid handling workstation 3 is equipped with a 96-channel first cleaning station 31 corresponding to the first pipette and a flexible 8-channel second cleaning station 34 corresponding to the second pipette, wherein the first cleaning station 31 is an independent structure with 96 holes; the second cleaning station 34 is an independent structure with 8 holes, and the independent water inlet pipeline and independent wastewater pipeline effectively avoid cross contamination.

[0150] f. Automated centrifugation, film sealing and film tearing: The first grasping structure in the automated nucleic acid aptamer screening system is a four-axis external robotic arm, which can realize the transportation of consumables between devices at different heights outside the workstation. The operation of the robotic arm and the series connection with the liquid handling workstation 3 and peripheral equipment are controlled by a controller, which can automatically realize the centrifugation, film sealing and film tearing of samples from the liquid handling workstation to the centrifuge 63, the film sealing machine 61 and the film tearing machine 62.

[0151] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above-mentioned technical features. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A nucleic acid aptamer automated screening system, characterized in that: include: A consumables stack (1), the consumables stack (1) is used to store reagents and consumables; A first transport track (2); A liquid handling workstation (3), the liquid handling workstation (3) is used for pipetting, mixing, dispensing and recovering liquid samples, and the first transport track (2) is used for transporting reagents or consumables stored in the consumables stack (1) to the liquid handling workstation (3); A second transport track (4); A first gripping structure (5), the first gripping structure (5) is used to transfer the liquid sample, and the second transport track (4) is used to transfer the liquid sample processed by the liquid processing workstation (3) to the gripping range of the first gripping structure (5); A sample processing and analysis module, which is used for processing liquid sample containers and analyzing liquid samples; A controller, the controller being electrically connected to the consumables stack (1), the first transport track (2), the liquid processing workstation (3), the second transport track (4), the first gripping structure (5) and the sample processing and analysis module; The sample processing and analysis module and the second transport track (4) are both located within the gripping range of the first gripping structure (5).

2. An automated nucleic acid aptamer screening system according to claim 1, characterized in that: Liquid handling workstation (3) includes: a first pipette (38); A first cleaning station (31), the first cleaning station (31) is used for cleaning the first pipette (38); A vortex oscillation module (32), the vortex oscillation module (32) is used for oscillating and mixing the liquid sample; A PCR module (33), wherein the PCR module (33) is used to perform a PCR reaction; a second pipette (39); A second cleaning station (34), the second cleaning station (34) is used for cleaning the second pipette (39); A garbage station (35), the garbage station (35) is used to collect waste generated by the first pipette (38) and / or the second pipette (39); A constant temperature incubator (36), the constant temperature incubator (36) is used to incubate the sample; Among them, the first pipette (38), the first cleaning station (31), the vortex oscillation module (32), the PCR module (33), the second pipette (39), the second cleaning station (34), the garbage station (35) and the constant temperature incubator (36) are all electrically connected to the controller.

3. A nucleic acid aptamer automated screening system according to claim 1, characterized in that: The liquid handling workstation (3) further comprises a second gripping structure (37) for pipetting and transporting consumables.

4. A nucleic acid aptamer automated screening system according to claim 1, characterized in that: The first pipette (38) is a 96-channel pipette, the first cleaning station (31) is a 96-channel cleaning station, the second pipette (39) is an 8-channel pipette, and the second cleaning station (34) is an 8-channel cleaning station.

5. A nucleic acid aptamer automated screening system according to claim 1, characterized in that: The second grasping structure (37) includes a first manipulator (371) and a second manipulator (372), the first pipette (38) is assembled on the first manipulator (371), and the second pipette (39) is assembled on the second manipulator (372).

6. A nucleic acid aptamer automated screening system according to claim 1, characterized in that: Sample processing and analysis modules include: A film sealing machine (61), the film sealing machine (61) is used to seal the container of the liquid sample; A film peeling machine (62), the film peeling machine (62) is used to remove the sealing film on the container of the liquid sample; A centrifuge (63), the centrifuge (63) is used to process a liquid sample; Sample analysis module, which is used for counterfeit analysis of intermediate products or final products in the screening process; The film sealing machine (61), the film tearing machine (62), the centrifuge (63) and the sample analysis module are all electrically connected to the controller.

7. An automated nucleic acid aptamer screening system according to claim 6, characterized in that: The sample analysis module includes: A fragment analyzer (641), wherein the fragment analyzer (641) is used for analyzing nucleic acid fragments during the nucleic acid aptamer screening process; A fluorescence quantitative PCR instrument (642), the fluorescence quantitative PCR instrument (642) is used to perform quantitative analysis on nucleic acids in a liquid sample; An enzyme-labeling instrument (643), the enzyme-labeling instrument (643) is used for performing quantitative and / or qualitative analysis on a liquid sample; Among them, the fragment analyzer (641), the fluorescence quantitative PCR instrument (642) and the microplate reader (643) are all electrically connected to the controller.

8. An automated nucleic acid aptamer screening system according to claim 1, characterized in that: The first grasping structure (5) is a four-axis robotic arm.

9. An automated nucleic acid aptamer screening system according to claim 1, characterized in that: A plurality of shelves and a rotating mechanism for driving the plurality of shelves to rotate are arranged in the consumable material stack (1); the rotating mechanism can drive the plurality of shelves to rotate, and the rotating mechanism is electrically connected to a controller.

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