Automatic screening method for nucleic acid aptamers

Abstract

The invention relates to a nucleic acid aptamer automatic screening method, and belongs to the technical field of nucleic acid aptamer screening, the method comprises the following steps: S1, preparing reagent consumables and an automatic workstation; s2, library denaturation and renaturation treatment; s3, reverse screening magnetic bead cleaning; s4, carrying out reverse screening on the magnetic beads and carrying out library incubation; s5, reverse screening and cleaning; s6, reverse screening elution product collection; s7, incubating the reverse screening supernatant and a protein tag; s8, washing and incubating positive screening magnetic beads; s9, performing positive screening and cleaning; s10, eluting a positive screening product; s11, collecting a positive screening elution product; s12, performing qPCR (quantitative polymerase chain reaction) detection on library enrichment conditions; s13, carrying out PCR (Polymerase Chain Reaction) amplification; s14, SA magnetic bead cleaning is carried out; s15, incubating a PCR product and SA magnetic beads; and S16, preparing an SA magnetic bead single chain. The invention provides an automatic screening method for nucleic acid aptamers, which can improve the reproducibility of experimental results and the experimental efficiency and improve the screening success rate.

Description

Technical Field

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

[0002] Nucleic acid aptamers are short single-stranded RNA or DNA oligonucleotide sequences. This sequence can form a partially complementary secondary structure, and further fold and entangle based on this to form a more complex three-dimensional spatial conformation. This conformation enables nucleic acid aptamers to recognize specific target molecules, which is similar to antibodies in function and is an important type of functional molecule. At the same time, nucleic acid aptamers have special properties such as nucleic acid amplification, sequencing and encoding, showing broad application prospects in many fields.

[0003] All aptamers are selected by in vitro evolution, a technique called systematic evolution of ligands by exponential enrichment (SELEX). The classic SELEX method usually covers three main steps: incubation, separation, and amplification. Specifically, first, the random library will naturally fold to form a variety of different types of secondary structures, including but not limited to hairpins, pseudoknots, protrusions, G-quadruplexes, etc., and then incubate and bind with the target. Next, different separation techniques are used to separate the unbound nucleic acid sequences to obtain sequences that bind to the target. Subsequently, the sequences that bind to the target are eluted and amplified by PCR amplification. Finally, the candidate sequences undergo multiple rounds of iterative screening, so that these sequences gradually dominate and enrich in the library, and then the screened sequences are sequenced and analyzed. However, the traditional SELEX method is usually cumbersome in the sample processing process and often relies on manual operations. It requires multiple rounds of screening to isolate aptamers with sufficient specificity and binding affinity, and it also has high technical operation requirements for experimenters. It lacks the ability to screen multiple targets with high throughput. It has defects such as low throughput, long time, high cost, high operation error rate, poor stability and low success rate, which seriously hinders the rapid development of this field.

[0004] With the widespread application of nucleic acid aptamers, the SELEX method is also constantly improving. In order to improve the screening efficiency and success rate, semi-automatic screening has gradually replaced traditional manual screening, such as a semi-automatic screening instrument based on a magnetic rod. The semi-automatic screening instrument is equipped with a matching deep-well plate and a magnetic rod sleeve. The magnetic rod sleeve is installed on the mobile device of the semi-automatic instrument and can be inserted into the deep-well plate to achieve mixing and magnetic suction operations. This semi-automatic screening method reduces some manual experimental steps, such as incubation and cleaning, and can screen more than a dozen targets at the same time. The screening efficiency is more than ten times higher than that of traditional manual screening, and the human interference factor is greatly reduced, saving a lot of time and manpower. However, the current semi-automatic screening method still has many shortcomings. On the one hand, some experimental operations still need to be performed manually, such as automated heating and elution, PCR amplification, and single-stranded production. In particular, the quality inspection parts such as qPCR detection, fragment analysis, and microplate reader concentration determination in the screening process monitoring are still unable to achieve automated operation. On the other hand, semi-automated screening can only automate some experimental steps, and the entire screening experimental step cannot be automated and run continuously, which still requires a lot of manpower and time. In addition, the throughput of semi-automated screening is not very high, and only a dozen targets can be screened in parallel, which has a great negative impact on the screening process and efficiency.

[0005] In view of the various shortcomings of existing technologies, there is an urgent need to develop new, more effective and faster SELEX methods to meet the growing needs of scientific research and applications. Summary of the invention

[0006] In order to overcome the above-mentioned defects of the prior art, the present invention provides an automated screening method for nucleic acid aptamers, which improves the reproducibility of experimental results and experimental efficiency and increases the screening success rate.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides a method for automated screening of nucleic acid aptamers, the method comprising:

[0008] S1: Preparation of reagents, consumables and automated workstations. Before automated screening, reagents, consumables and instruments must be prepared. The automated workstation includes instruments and equipment, and the preparation of the automated workstation includes initialization of instruments and equipment.

[0009] S2: library denaturation and renaturation treatment, the single-stranded DNA random library is denatured and renatured by an automated workstation;

[0010] S3: Counter-screening magnetic bead washing: automatic counter-screening magnetic bead washing is performed through an automated workstation, the washing solution is aspirated into a counter-screening magnetic bead deep-well plate, and after oscillation, the supernatant is aspirated and transferred to the washing station to complete the counter-screening magnetic bead washing;

[0011] S4: Incubation of the reverse screening magnetic beads and the library. The reverse screening magnetic beads and the library are incubated automatically in the automated workstation. The renatured library is transferred to the reverse screening magnetic bead deep-well plate. After processing, the reverse screening magnetic beads and the library are incubated.

[0012] S5: Back-screen cleaning, automatically control the automated workstation to perform automated back-screen cleaning;

[0013] S6: collecting the back-screening elution product, collecting the back-screening elution product through an automated workstation;

[0014] S7: Incubate the supernatant with the protein label. Use an automated workstation to seal the deep-well plate containing the supernatant and the protein label, and then fully shake and incubate.

[0015] S8: Positive screening magnetic bead washing and incubation, automatic control of the automated workstation for automated positive screening magnetic bead washing and positive screening magnetic bead incubation with the library;

[0016] S9: Main screen cleaning, automatic main screen cleaning is performed through an automated workstation;

[0017] S11: positive screen elution product collection, automatic positive screen elution product collection is performed through an automated workstation;

[0018] S12: qPCR detection of library enrichment, qPCR detection is performed by an automated workstation;

[0019] S13: PCR amplification: automatic PCR amplification is performed by an automated workstation, and the eluted product of the positive screening is transferred to a PCRMix plate for PCR amplification;

[0020] S14: SA magnetic bead washing: automatic SA magnetic bead washing is performed through an automated workstation, the washing solution is aspirated into the SA magnetic bead deep-well plate, and the supernatant in the SA magnetic bead deep-well plate is aspirated after treatment, and transferred to the washing station to complete the SA magnetic bead washing;

[0021] S15: Incubation of PCR products with SA magnetic beads is performed by an automated workstation to complete the incubation of PCR products with SA magnetic beads;

[0022] S16: SA magnetic bead single-strand preparation, which is completed by operating on an automated workstation.

[0023] Furthermore, in step S2, when the single-stranded DNA random library is subjected to denaturation and renaturation treatment by an automated workstation, in the automated workstation, the DPBS buffer is first aspirated and transferred to the well plate containing the random library, and then placed in an oscillation module for sufficient oscillation. After sufficient oscillation, the single-stranded DNA random library is aspirated therefrom and transferred to a PCR plate, and then sealed with a film. After sealing with a film, the single-stranded DNA random library is transferred to a PCR instrument for denaturation and renaturation treatment.

[0024] Furthermore, in step S5, automated back-screening cleaning is performed by automatically controlling an automated workstation. In the automated workstation, the back-screening sample deep-well plate is transferred to the magnetic stand Mag and allowed to stand for magnetic attraction. The supernatant of the back-screening sample is transferred to the deep-well plate containing the protein label, and then the DPBS-T washing solution is transferred to the back-screening magnetic bead deep-well plate. After sufficient oscillation, the plate is allowed to stand for magnetic attraction. The supernatant is transferred to the cleaning station to complete the back-screening cleaning.

[0025] Furthermore, in step S6, the back screening elution product is collected by an automated workstation, and in the automated workstation, the eluted back screening magnetic bead sample deep well plate is transferred to the magnetic stand Mag for static magnetic absorption, and the back screening sample supernatant is sucked and transferred to the deep well plate, which is recorded as the back screening elution sample Elution-;

[0026] In the step S11, the positive screen elution product is automatically collected by an automated workstation, the eluted positive screen magnetic bead sample deep well plate is transferred to the magnetic stand Mag for static magnetic absorption, and the positive screen sample supernatant is transferred to the deep well plate, which is recorded as the positive screen elution sample Elution+.

[0027] Furthermore, in step S8, the automatic positive screening magnetic beads are automatically cleaned and the positive screening magnetic beads are incubated with the library by automatically controlling the automated workstation. In the automated workstation, DPBS-T washing solution is added to the positive screening magnetic beads deep well plate, and then the plate is transferred to the oscillation module for sufficient oscillation, and then transferred to the magnetic stand Mag for static magnetic absorption, the supernatant of the positive screening magnetic beads deep well plate is transferred to the cleaning station, and the positive screening magnetic beads deep well plate is transferred to the constant temperature incubator to complete the positive screening magnetic beads cleaning; subsequently, the sample containing the reverse screening supernatant and the protein label is transferred to the positive screening magnetic beads deep well plate, and the plate is fully oscillated and incubated in the oscillation module to complete the incubation of the positive screening magnetic beads with the library.

[0028] Furthermore, in step S9, automated positive screen cleaning is performed by an automated workstation. In the automated workstation, DPBS-T washing solution is added to the positive screen magnetic bead deep well plate, which is transferred to an oscillation module for sufficient oscillation. After magnetic attraction, the supernatant is aspirated and transferred to the deep well plate, recorded as Wash1+, to complete the first positive screen cleaning. The above steps are repeated, and after magnetic attraction, the supernatant is aspirated and transferred to another deep well plate, recorded as Wash2+, to complete the second positive screen cleaning.

[0029] Furthermore, in step S12, an automated workstation is used to perform the step, in which the qPCR Mix in the constant temperature incubator is transferred to the 384 wells of the 384qPCR1 plate, and Wash1+, Wash2+, Elution+, and Elution- are respectively aspirated and transferred to the 384qPCR1 plate, and the 384qPCR1 plate is placed in a sealing machine and sealed before being transferred for qPCR detection; the above steps are repeated to complete the parallel qPCR detection of the 384qPCR2 plate.

[0030] Furthermore, in step S15, the operation is performed through an automated workstation, in which the samples in the PCR1 plate to the PCR6 plate are first aspirated and transferred to deep-well plates containing SA magnetic beads and NaCl, respectively, and then sealed. After sealing, the plates are transferred to an oscillation module, and the PCR products and the SA magnetic beads are fully oscillated and incubated.

[0031] Furthermore, in step S16, the operation is performed through an automated workstation, in which the SA magnetic beads are first cleaned after incubation, and then alkaline elution is performed, and the eluted supernatant is transferred to a deep-well plate for collecting ssDNA products, and HCl and 2×DPBS are aspirated and transferred to the deep-well plate of ssDNA products to complete the single-stranded preparation.

[0032] Further, the automated workstation includes a rack, on which a consumables stacking area, a liquid processing workstation, a first transverse transport track for transporting consumables, a second longitudinal transport track for transporting consumables, and a first grabbing structure are provided, the consumables stacking area is arranged on one side of the liquid processing workstation, and the first grabbing structure is located at the rear of the liquid processing workstation;

[0033] The liquid handling workstation includes a first cleaning station, a vortex oscillation module, a PCR module, a second cleaning station, a garbage station, a constant temperature incubator, a second gripping structure and a pipette, wherein the second gripping structure includes a first manipulator and a second manipulator;

[0034] The frame is also provided with a film sealing machine, a film tearing machine, a centrifuge, a fragment analyzer, a fluorescence quantitative PCR instrument, and an enzyme marker. The film sealing machine, the film tearing machine, and the centrifuge are all arranged on the right side of the first grasping structure, and the fragment analyzer, the fluorescence quantitative PCR instrument, and the enzyme marker are arranged on the left side of the first grasping structure.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The operation of the entire method is controlled by the system control program, and can be run quickly and accurately according to the set program. The method set by the system program can automatically complete all experimental operations in one round of screening, and each experimental link can be automatically connected. Therefore, you only need to prepare samples and plans and run the method of the present invention to obtain a round of screening products. Through multiple rounds of automated screening, nucleic acid aptamers for multiple targets are finally obtained;

[0037] The present invention provides a method for automated screening of nucleic acid aptamers, which can process the number of targets corresponding to the number of holes simultaneously according to the number of holes of a multi-well plate in a liquid handling workstation, greatly improving the screening efficiency. Traditional single-target screening requires 2-3 weeks, while this method can complete the screening of the number of targets corresponding to the number of holes within 5-7 days. The entire method is controlled by a system control program, and can be run quickly and accurately according to the set method program, which minimizes human intervention and greatly improves the reproducibility and experimental efficiency of the experimental results. This method can expand the experimental design throughput, avoid human errors, accelerate research progress, accelerate the acquisition of research results, and is conducive to shortening the research and development cycle of new nucleic acid aptamers on the basis of an automated screening system. The SELEX method in the prior art needs to rely on manual implementation, and there are shortcomings such as low throughput, long time consumption, high cost, high operating error rate, poor stability, and low success rate. In particular, the quality inspection parts such as qPCR detection, fragment analysis, and microplate reader concentration determination in the screening process monitoring realize automated operation, which improves the screening success rate, and is a more effective and faster new SELEX method. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a flowchart of the screening method of the present invention;

[0039] Figure 2 It is a structural schematic diagram of an automated workstation according to an embodiment of the present invention;

[0040] Figure 3 It is a schematic diagram of the three-dimensional structure of a liquid processing workstation according to an embodiment of the present invention;

[0041] Figure 4 This is a schematic diagram of the structure of a liquid processing workstation table according to an embodiment of the present invention;

[0042] Figure 5 This is a schematic diagram of the table layout of a liquid processing workstation according to Example 2 of the present invention;

[0043] Figure 6 This is a schematic diagram of the table layout of a liquid processing workstation according to Example 3 of the present invention;

[0044] Figure 7 It is a schematic diagram of the table layout of the liquid processing workstation according to Example 4 of the present invention;

[0045] Figure 8 This is a schematic diagram of the table layout of a liquid processing workstation according to Example 5 of the present invention;

[0046] Fig. 9 This is a schematic diagram of the table layout of a liquid processing workstation according to Example 6 of the present invention;

[0047] Fig.10This is a schematic diagram of the table layout of a liquid processing workstation according to Example 7 of the present invention;

[0048] Fig.11 This is a schematic diagram of the table layout of the liquid processing workstation in Example 8 of the present invention;

[0049] Fig.12 This is a schematic diagram of the table layout of a liquid processing workstation according to Example 9 of the present invention;

[0050] Fig.13 It is a schematic diagram of the table layout of the liquid processing workstation according to embodiment 10 of the present invention;

[0051] Fig.14 This is a schematic diagram of the table layout of the liquid processing workstation in Example 11 of the present invention;

[0052] Fig.15 This is a schematic diagram of the table layout of a liquid processing workstation according to Example 12 of the present invention;

[0053] Fig.16 This is a schematic diagram of the table layout of the liquid processing workstation in Example 13 of the present invention;

[0054] Fig.17 This is a schematic diagram of the table layout of a liquid processing workstation according to Example 14 of the present invention;

[0055] Fig.18 This is a schematic diagram of the table layout of the liquid processing workstation in Example 15 of the present invention;

[0056] Fig.19 This is a schematic diagram of the table layout of the liquid processing workstation in Example 16 of the present invention;

[0057] Fig. 20 Schematic diagram of the Cq value of the positive sieve elution product (E+) of an embodiment of the present invention;

[0058] Fig.21 Schematic diagram of the Cq value of the back-screening elution product (E-) in an embodiment of the present invention;

[0059] Fig. 22 This is a schematic diagram of the verification results of the single-stranded product of the present invention;

[0060] Fig.23 This is a schematic diagram of the quality verification results of the single-stranded product of the present invention;

[0061] Fig.24 Schematic diagram of the affinity of the successful nucleic acid aptamers screened for the embodiments of the present invention. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the invention, rather than all the embodiments. The embodiments of the present invention are described below in conjunction with the drawings.

[0063] Example 1

[0064] refer to Figure 1-19 , a method for automated screening of nucleic acid aptamers, comprising the following steps:

[0065] S1: Preparation of reagents and consumables, and automated workstation. Before automated screening, reagents and consumables, and instruments and equipment must be prepared. The consumables include: 90μL pipette tips, 230μL pipette tips, deep-well plates, reagent tanks, PCR plates, and qPCR plates. The automated workstation includes instruments and equipment. The preparation of the automated workstation includes initialization of instruments and equipment.

[0066] S2: library denaturation and renaturation treatment: the single-stranded DNA random library is denaturated and renatured by an automated workstation, the DPBS buffer is transferred to the well plate containing the random library, and the well plate is placed in an oscillation module for full oscillation. After full oscillation, the ssDNA library is transferred to the PCR plate, sealed with a film, and then transferred to a PCR instrument for denaturation and renaturation treatment;

[0067] S3: Counter-screening magnetic bead washing: automatic counter-screening magnetic bead washing is performed through an automated workstation. DPBS-T washing solution is aspirated into a counter-screening magnetic bead deep-well plate, placed in an oscillating module for full oscillation, and then placed on a magnetic stand Mag for static magnetic absorption. The supernatant is aspirated and transferred to the washing station to complete the counter-screening magnetic bead washing.

[0068] S4: Incubate the anti-screening magnetic beads with the library. The anti-screening magnetic beads and the library are incubated automatically in the automated workstation. The renatured library is transferred to the anti-screening magnetic bead deep-well plate. The anti-screening magnetic beads and the library are fully incubated in the oscillation module to complete the incubation.

[0069] S5: Back-screening cleaning: the automated workstation is automatically controlled to perform automated back-screening cleaning. The back-screening sample deep-well plate is transferred to the magnetic stand Mag and placed in a static magnetic absorption state. The supernatant of the back-screening sample is transferred to the deep-well plate containing the protein label, and then the DPBS-T washing solution is transferred to the back-screening magnetic bead deep-well plate. After sufficient oscillation, the plate is placed in a static magnetic absorption state, and the supernatant is transferred to the cleaning station to complete the back-screening cleaning.

[0070] S6: Collect the eluted products of the back-screening. The eluted products of the back-screening magnetic beads are collected by the automated workstation. The eluted back-screening magnetic beads sample deep-well plate is transferred to the magnetic stand Mag for static magnetic absorption. The supernatant of the back-screening sample is aspirated and transferred to the deep-well plate, which is recorded as the back-screening elution sample Elution-;

[0071] S7: Incubate the supernatant and protein label. Move the deep-well plate containing the supernatant and protein label to the sealing machine through the automated workstation for automatic sealing. After sealing, move the plate to the shaking module for full shaking and incubation.

[0072] S8: Positive screening magnetic bead washing and incubation, automatic control of the automated workstation for automated positive screening magnetic bead washing, and incubation of the positive screening magnetic beads with the library; DPBS-T washing solution is added to the positive screening magnetic bead deep-well plate, and then transferred to the oscillation module for sufficient oscillation, and then transferred to the magnetic stand Mag for static magnetic absorption, the supernatant of the positive screening magnetic bead deep-well plate is transferred to the washing station, and the positive screening magnetic bead deep-well plate is transferred to the constant temperature incubator to complete the positive screening magnetic bead washing; then, the sample containing the reverse screening supernatant and protein label is transferred to the positive screening magnetic bead deep-well plate, and fully oscillated and incubated in the oscillation module to complete the incubation of the positive screening magnetic beads with the library;

[0073] S9: Positive screen washing: perform automatic positive screen washing through an automated workstation, add DPBS-T washing solution to the positive screen magnetic bead deep-well plate, transfer to the shaking module for full shaking, and transfer the supernatant to the deep-well plate after magnetic suction, which is recorded as Wash1+, completing the first positive screen washing. Repeat the above steps, transfer the supernatant to another deep-well plate after magnetic suction, which is recorded as Wash2+, completing the second positive screen washing;

[0074] S10: Elution of positive screening products: automatic elution of positive screening products is performed through an automated workstation;

[0075] S11: Collection of positive screen elution products: The positive screen elution products are automatically collected through an automated workstation. The eluted positive screen magnetic bead sample deep-well plate is transferred to the magnetic stand Mag for static magnetic absorption. The positive screen sample supernatant is aspirated and transferred to the deep-well plate, which is recorded as the positive screen elution sample Elution+;

[0076] S12: qPCR detection of library enrichment is performed on an automated workstation. The qPCRMix in the constant temperature incubator is transferred to the 384 wells of the 384qPCR1 plate. Wash1+, Wash2+, Elution+, and Elution- are respectively transferred to the 384qPCR1 plate. The 384qPCR1 plate is sealed with a film sealing machine and then transferred for qPCR detection. The above steps are repeated to complete the parallel qPCR detection of the 384qPCR2 plate.

[0077] S13: PCR amplification: automatic PCR amplification is performed through an automated workstation, Elution+ is transferred to the PCR Mix deep-well plate, and after mixing, the plates are transferred to PCR1 to PCR6 respectively, and then the plates are sealed with films for PCR amplification;

[0078] S14: SA magnetic bead washing, aspirate DPBS-T washing solution and transfer it to an empty deep-well plate, then transfer it to the SA magnetic bead deep-well plate, then transfer it to the shaking module for full shaking, transfer it to the magnetic stand Mag for static magnetic absorption, aspirate the supernatant in the SA magnetic bead deep-well plate, and transfer it to the washing station to complete the SA magnetic bead washing;

[0079] S15: Incubate PCR products with SA magnetic beads. The automated workstation is used to transfer samples from PCR1 to PCR6 plates to deep-well plates containing SA magnetic beads and NaCl, and then seal the plates. After sealing, the plates are transferred to the oscillation module, and the PCR products and SA magnetic beads are fully oscillated and incubated.

[0080] S16: SA magnetic bead single-strand preparation, operated by an automated workstation, first wash the SA magnetic beads after incubation, then perform alkaline elution, transfer the eluted supernatant to a deep-well plate for collecting ssDNA, and absorb HCl and 2×DPBS and transfer to the deep-well plate for ssDNA to complete the single-strand preparation;

[0081] The automated workstation in the method includes a rack, on which are provided a consumables stacking area 1, a liquid handling workstation 3, a first transverse transport track 2 for transporting consumables, a second longitudinal transport track 4 for transporting consumables, and a first gripping structure 5. The liquid handling workstation 3 includes a first cleaning station 31, a vortex oscillation module 32, a PCR module 33, a second cleaning station 34, a garbage station 35, a constant temperature incubator 36, a second gripping structure 37 and a pipette 38. The second gripping structure 37 includes a first manipulator 371 and a second manipulator 372. The second manipulator 372, the first transport track 2 is used to transport consumables from the consumables stacking area 1 to the liquid processing workstation 3. The second manipulator 372 grabs the consumables from the liquid processing workstation 3 and sends them along the second transport track 4 to the grabbing range of the first grabbing structure 5, and then sends them to the film sealing machine 61 for film sealing and other treatments, and to the fluorescent quantitative PCR instrument 642 for detection. The first manipulator 371 sends the consumables from the consumables stacking area 1 to the liquid processing workstation 3 along the first transport track 2. When in use, the steps of the above method are automatically controlled to complete;

[0082] The frame is also provided with a film sealing machine 61, a film tearing machine 62, a centrifuge 63, a fragment analyzer 641, a fluorescence quantitative PCR instrument 642, and an enzyme marker 643; the first grasping structure 5 is arranged at the rear of the liquid handling workstation 3, the film sealing machine 61, the film tearing machine 62, and the centrifuge 63 are all arranged on the right side of the first grasping structure 5, and the fragment analyzer 641, the fluorescence quantitative PCR instrument 642, and the enzyme marker 643 are arranged on the left side of the first grasping structure 5, so as to facilitate the grasping of the first grasping structure 5. After grasping, the film sealing machine 61, the film tearing machine 62, and the centrifuge 63 can be placed for sealing, tearing and centrifugation, and then grasped to the fragment analyzer 641, the fluorescence quantitative PCR instrument 642, and the enzyme marker 643 for analysis.

[0083] Example 2

[0084] Automated single-stranded DNA (ssDNA) random library renaturation method, reference Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place the BC230 gun tip at the TL6 position, the 96-well plate containing the random library at the P8 position, and the 96PCR plate at the P7 position;

[0085] Specific operation steps: the first pipette 381 inserts the 96BC230 tip at the TL6 position, absorbs 100 μL of DPBS buffer at the P2 position, and transfers it to the library at the P8 position; the first manipulator 371 grabs the 96-well plate at the P8 position and places it in the oscillation module 32, oscillates it at 1000 rpm for 1 minute, and the first manipulator 371 grabs the 96-well plate at the oscillation module 32 position and places it at the P8 position; the first pipette 381 absorbs 100 μL of the ssDNA library at the P8 position and transfers it to the 96PCR plate at the P7 position; the first manipulator 371 grabs the The 96-well PCR plate is placed at the P24 position and transported to the grabbing range of the first grabbing structure 5 through the second transport track 4; the first grabbing structure 5 grabs the 96-well PCR plate and places it in the sealing machine 61 for PCR plate sealing; after sealing, the 96-well PCR plate is transferred to the P7 position through the first grabbing structure 5, the second transport track 4 and the first manipulator 371; finally, the first manipulator 371 grabs the 96-well plate at the P7 position and places it in the PCR instrument 33ATC1, and the system controls the PCR instrument to set the program to 95℃10min, 4℃5min, and 25℃2h for denaturation treatment.

[0086] Example 3

[0087] Automated reverse screening magnetic bead cleaning, reference Figure 2 , Figure 3 , Figure 4 , Figure 6, the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place BC230 gun tips at positions TL2 and TL3 respectively, place a reagent tank containing 10mL screening pressure buffer (containing BSA with a final concentration of 0.1mg / mL, HsDNA with a final concentration of 0.1mg / mL, and Mg2+ with a final concentration of 10mM) at position P12, place a deep-well plate containing 50μL reverse screening magnetic beads at position 36 of the constant temperature incubator, place a deep-well plate containing 1mL DPBS-T washing solution at position P8, and place a magnetic stand Mag at position P9;

[0088] Specific operation steps: the first pipette 381 inserts the 96BC230 tip at the TL2 position, absorbs 200 μL of DPBS-T washing solution at the P8 position, and transfers it to the reverse screening magnetic bead deep well plate at the constant temperature incubator 36 position; the first manipulator 371 grabs the deep well plate at the constant temperature incubator 36 position and places it in the oscillation module 32, and oscillates it at 1000 rpm for 1 minute; the first manipulator 371 grabs the deep well plate of the oscillation module 32 and places it at the P6 position; the first manipulator 371 grabs the deep well plate at the P6 position and places it on the magnetic stand Mag at the P9 position, and allows it to stand for 2 minutes; the first pipette 381 absorbs all the supernatant in the deep well plate at the P9 position, transfers it to the first cleaning station 31, and cleans the tip on the first pipette 381 here; the first manipulator 371 grabs the deep well plate at the P9 position and places it in the constant temperature incubator 36 position. At this point, one reverse screening magnetic bead cleaning is completed;

[0089] Repeat the above experimental operation twice again to complete three washes of the counter-screening magnetic beads; then, the first pipette 381 puts the 96BC230 tip back to the TL2 position; the first pipette 381 inserts the 96BC230 tip at the TL7 position, absorbs 100 μL of screening pressure buffer at the P12 position, and transfers it to the counter-screening magnetic bead deep-well plate at the constant temperature incubator 36 position; the first pipette 381 puts the 96BC230 tip back to the TL7 position; the first manipulator 371 grabs the deep-well plate at the constant temperature incubator 36 position and places it in the oscillation module 32, and oscillates it at 1000 rpm for 30 minutes; the first manipulator 371 grabs the deep-well plate of the oscillation module 32 and places it in the constant temperature incubator 36 position.

[0090] Example 4

[0091] Automated counter-screening magnetic beads and library incubation, reference Figure 2 , Figure 3 , Figure 4 , Figure 7, the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; according to Example 2, the gun tips and samples at the positions of TL7 and the constant temperature incubator 36 in this embodiment are consistent; according to Example 1, the position P4 in this embodiment is a library with good renaturation (96PCR plate);

[0092] Specific operation steps:

[0093] Film tearing: The first manipulator 371 transfers the renatured library at position P4 to position P24, and transmits it to the grabbing range of the first grabbing structure 5 through the second transport track 4; the first grabbing structure 5 grabs the deep-well plate and places it in the film tearing machine 62 for film tearing; after the film tearing is completed, the library PCR plate is transferred to position P4 through the first grabbing structure 5, the second transport track 4 and the first manipulator 371;

[0094] The first pipette 381 inserts the 96BC230 tip at the TL7 position, absorbs 100 μL of the renatured library at the P4 position, and transfers it to the reverse screening magnetic bead deep well plate at the constant temperature incubator 36 position; the first pipette 381 puts the 96BC230 tip back to the TL7 position; the first manipulator 371 grabs the deep well plate at the constant temperature incubator 36 position and places it in the oscillation module 32, and oscillates it at 1000 rpm for 30 minutes; the first manipulator 371 grabs the deep well plate of the oscillation module 32 and places it in the constant temperature incubator 36 position.

[0095] Example 5

[0096] Automatic back screen cleaning, reference Figure 2 , Figure 3 , Figure 4 , Figure 8 , the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place BC230 gun tips at positions TL2, TL3, TL6 and TL7 respectively; place the reverse screening sample deep well plate on the constant temperature incubator 36 in Example 3 at position P3, place a 96 deep well plate containing 4 μL of 5 mg / mL protein label in each well at the constant temperature incubator 36 of this embodiment, place a deep well plate containing 1 mL of DPBS-T washing solution at position P4, place a magnetic stand Mag at position P9, place an empty deep well plate for reverse screening elution at position P10, and place a deep well plate containing 300 μL of H2O at position P14;

[0097] The specific operation steps are as follows: the first manipulator 371 grabs the reverse screening sample deep-well plate at the P3 position and transfers it to the magnetic stand Mag at the P9 position, and allows it to stand for 2 minutes; the first pipette 381 inserts the 96BC230 gun tip at the TL6 position, absorbs all the reverse screening sample supernatant in the deep-well plate on the magnetic stand at the P9 position, and transfers it to the deep-well plate containing the protein label at the constant temperature incubator 36 position; the first pipette 381 puts the 96BC230 gun tip back to the TL6 position; the first manipulator 371 grabs the reverse screening magnetic bead deep-well plate on the magnetic stand Mag at the P9 position and places it at the P6 position;

[0098] First reverse screening cleaning: The first pipette 381 inserts the 96BC230 tip at the TL2 position, absorbs 200 μL of DPBS-T washing solution at the P4 position and transfers it to the reverse screening magnetic bead deep-well plate at the P6 position. The first pipette 381 puts the 96BC230 tip back to the TL2 position; the first manipulator 371 grabs the deep-well plate at the P6 position and places it in the oscillation module 32, oscillating it at 1000 rpm for 1 min; the first manipulator 371 grabs the deep-well plate at the oscillation module 32 and places it in the P6 position. The hand 371 grabs the deep-well plate at the P6 position and places it on the magnetic rack Mag at the P9 position, and allows it to stand for magnetic attraction for 1 minute; the first pipette 381 inserts the 96BC230 gun tip at the TL2 position, absorbs all the supernatant in the deep-well plate on the magnetic rack at the P9 position, and transfers it to the first cleaning station 31; the first pipette 381 puts the 96BC230 gun tip back to the TL2 position, and the first manipulator 371 grabs the reverse screening magnetic bead deep-well plate on the magnetic rack Mag at the P9 position and places it at the P6 position; at this point, the first reverse screening cleaning is completed;

[0099] Second, third, and fourth reverse sieve cleaning: The first pipette 381 inserts the 96BC230 tip at the TL3 position, repeats the above cleaning operation 3 times, and completes 3 reverse sieve cleanings; the first pipette 381 puts the 96BC230 tip back to the TL3 position. So far, a total of 4 reverse sieve cleanings have been completed;

[0100] The first pipette 381 inserts the 96BC230 tip at the TL7 position, and transfers 100 μL of H2O in the deep-well plate at the P14 position to the reverse screening magnetic bead deep-well plate at the P6 position. The first manipulator 371 grabs the reverse screening magnetic bead deep-well plate at the P6 position and places it in the oscillation module 32, and oscillates it at 1000 rpm for 1 min. The first manipulator 371 grabs the deep-well plate of the oscillation module 32 and places it at the P6 position. The first pipette 381 transfers 100 μL of mixed magnetic beads in the deep-well plate at the P6 position to the deep-well plate at the P10 position. The first pipette again transfers 100 μL of H2O in the deep-well plate at the P14 position. H2O is transferred to the counter-screening magnetic bead deep-well plate at the P6 position, the first manipulator 371 grabs the counter-screening magnetic bead deep-well plate at the P6 position and places it in the oscillation module 32, and oscillates it at 1000 rpm for 1 min; the first manipulator 371 grabs the deep-well plate of the oscillation module 32 and places it at the P6 position, the first pipette 381 absorbs 100 μL of mixed magnetic beads in the deep-well plate at the P6 position and transfers it to the deep-well plate at the P10 position; the first pipette 381 puts the 96BC230 gun tip back to the TL7 position; the first manipulator 371 grabs the deep-well plate at the P6 position and places it at the P3 position.

[0101] Example 6

[0102] Automated back screening elution product collection, reference Figure 2 , Figure 3 , Figure 4 , Fig. 9 , the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place the BC230 gun head at the TL6 position; place the deep well plate for collecting the back-screening elution sample at the P4 position; place the back-screening magnetic bead sample at the P10 position, which is consistent with the P10 position in Example 4; place the magnetic stand Mag at the P9 position;

[0103] The specific operation steps are as follows: the deep well plate at position P10 is placed in the film sealing machine 61 for film sealing by the first manipulator 371, the second transport track 4, and the first grabbing structure 5; after film sealing, the deep well plate of the reverse screening magnetic bead sample is transferred to the constant temperature incubator 36 by the first grabbing structure 5, the second transport track 4, and the first manipulator 371, and heated at 100°C for 10 minutes; after heating and eluting, the deep well plate at position P10 is placed in the film tearing machine 62 for film tearing by the first manipulator 371, the second transport track 4, and the first grabbing structure 5; after film tearing, the deep well plate is placed in the film tearing machine 62 by the first grabbing structure 5, the second transport track 4, and the first grabbing structure 5 Transport track 4, the first manipulator 371 transfers the reverse screening magnetic bead sample deep well plate to the magnetic stand Mag at position P9, and leaves it to stand for magnetic attraction for 2 minutes; the first pipette 381 inserts the 96BC230 gun tip at position TL6, absorbs all the reverse screening sample supernatant in the deep well plate on the magnetic stand at position P9, and transfers it to the deep well plate at position P4, which is the reverse screening elution sample Elution-; the first pipette 381 puts the 96BC230 gun tip back to position TL6; the first manipulator 371 grabs the reverse screening magnetic bead deep well plate on the magnetic stand Mag at position P9 and places it at position P10.

[0104] Example 7

[0105] Automated counter-screening supernatant incubated with protein label, reference Figure 2 , Figure 3 , Figure 4 , Fig.10 After the back-screening elution is completed, according to the instructions set by the SAMI system program, the deep-well plate containing the back-screening supernatant and the protein label in Example 4 is transferred to the P24 position through the first transport track 2 and the first manipulator 371, and is transmitted to the grasping range of the first grasping structure 5 through the second transport track 4; the first grasping structure 5 grasps the deep-well plate and places it in the film sealing machine 61 for film sealing; after the film sealing is completed, the deep-well plate is transferred to the oscillation module 32 through the first grasping structure 5, the second transport track 4 and the first manipulator 371, and is fully oscillated at 1000rpm for 30 minutes.

[0106] Example 8

[0107] Automated positive screening magnetic bead washing and incubation, reference Figure 2 , Figure 3 , Figure 4 , Fig.11, the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place the BC230 gun tip at the TL2 and TL6 positions respectively, place the deep-well plate containing the reverse screening supernatant and protein label in Example 6 at the P4 position, place the deep-well plate containing 100 μL of positive screening magnetic beads at the constant temperature incubator 36 position, place an empty 96 deep-well plate at the P8 position, place the deep-well plate containing 1 mL of DPBS-T washing solution at the P11 position, and place the magnetic stand Mag at the P9 position;

[0108] The specific operation steps are: the first pipette 381 inserts the 96BC230 tip at the TL2 position, absorbs 200 μL of DPBS-T washing solution at the P11 position, and transfers it to the positive screening magnetic bead deep well plate at the constant temperature incubator 36 position; the first manipulator 371 grabs the deep well plate at the constant temperature incubator 36 position and places it in the oscillation module 32, and oscillates it at 1000 rpm for 1 minute; the first manipulator 371 grabs the deep well plate of the oscillation module 32 and places it at the P6 position; the first manipulator 371 grabs the deep well plate at the P6 position and places it on the magnetic stand Mag at the P9 position, and allows it to stand for magnetic attraction for 2 minutes; the first pipette 381 absorbs all the supernatant in the deep well plate at the P9 position, transfers it to the first cleaning station 31, and cleans the tip on the first pipette 381 here; the first manipulator 371 grabs the deep well plate at the P9 position and places it at the constant temperature incubator 36 position. At this point, the positive screening magnetic beads are washed once, and the above experimental operation is repeated 2 more times to complete the 3rd washing of the positive screening magnetic beads;

[0109] Subsequently, the first pipette 381 sucked 200 μL of DPBS-T washing solution at the P11 position; transferred it to the positive screening magnetic bead deep-well plate at the constant temperature incubator 36 position; the first manipulator 371 grabbed the deep-well plate at the constant temperature incubator 36 position and placed it in the oscillation module 32, and oscillated it at 1000 rpm for 1 minute; the first manipulator 371 grabbed the deep-well plate of the oscillation module 32 and placed it at the P6 position, and the first pipette 381 sucked 100 μL of the positive screening magnetic beads at the P6 position and transferred it to the deep-well plate at the P8 position; The robot 371 grabs the positive screening magnetic bead deep well plate at the P6 position and places it at the constant temperature incubator 36 position; the first robot 371 grabs the positive screening magnetic bead deep well plate at the P8 position and places it at the P9 magnetic stand Mag position, and allows it to stand for 2 minutes; the first pipette 381 absorbs all the supernatant in the deep well plate at the P9 position and transfers it to the first cleaning station 31, and the first robot 371 grabs the positive screening magnetic bead deep well plate on the magnetic stand at the P9 position and places it at the P6 position; the first pipette 381 puts the 96BC230 gun head back to the TL2 position;

[0110] Film tearing: The deep-well plate containing the supernatant and the protein label at the P4 position is transferred to the P24 position by the first manipulator 371, and is transported to the grabbing range of the first grabbing structure 5 by the second transport track 4; the first grabbing structure 5 grabs the deep-well plate and places it in the film tearing machine 62 for film tearing; after the film tearing is completed, the deep-well plate is transferred to the P4 position by the first grabbing structure 5, the second transport track 4 and the first manipulator 371;

[0111] The first pipette 381 inserts the 96BC230 tip at the TL6 position, and draws 200 μL of the sample containing the reverse screening supernatant and protein label at the P4 position and places it in the positive screening magnetic bead deep well plate at the P6 position; according to Example 6, the positive screening incubation sample at the P6 position is sealed with a film, and then placed back to the original P6 position after sealing. Then the first manipulator 371 grabs the deep well plate at the P6 position and places it in the oscillation module 32, oscillating it at 1000 rpm for 60 minutes to perform positive screening incubation. After the incubation is completed, the first manipulator 371 grabs the deep well plate at the oscillation module 32 position and places it at the P8 position.

[0112] Example 9

[0113] Automatic front screen cleaning, reference Figure 2 , Figure 3 , Figure 4 , Fig.12 , the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place BC230 gun tips at positions TL2, TL3, TL6 and TL7 respectively; place the sample deep-well plate after positive screen incubation at position P3, which is consistent with the position P8 in Example 7; place an empty deep-well plate for the supernatant after positive screen incubation at position P4, place a deep-well plate for the first supernatant of positive screen washing at position P8, place a deep-well plate for the second supernatant of positive screen washing at position P11, place a deep-well plate containing 1mL DPBS-T washing solution at position P12, place a magnetic stand Mag at position P9, place an empty deep-well plate for positive screen elution at position P10, and place a deep-well plate containing 300μL H2O at position P14;

[0114] The specific operation steps are as follows: the first manipulator 371 grabs the positive screening sample deep-well plate at the P3 position and transfers it to the magnetic stand Mag at the P9 position, and allows it to stand for 2 minutes; the first pipette 381 inserts the 96BC230 gun head at the TL6 position, absorbs all the positive screening sample supernatant in the deep-well plate on the magnetic stand at the P9 position, and transfers it to the deep-well plate at the P4 position; the first pipette 381 puts the 96BC230 gun head back to the TL6 position; the first manipulator 371 grabs the positive screening magnetic bead deep-well plate on the magnetic stand Mag at the P9 position and places it at the P6 position;

[0115] First positive screen cleaning: The first pipette 381 inserts the 96BC230 tip at the TL2 position, absorbs 200 μL of DPBS-T washing solution at the P12 position and transfers it to the positive screen magnetic bead deep-well plate at the P6 position. The first pipette 381 puts the 96BC230 tip back to the TL2 position; the first manipulator 371 grabs the deep-well plate at the P6 position and places it in the oscillation module 32, oscillating it at 1000 rpm for 1 min; the first manipulator 371 grabs the deep-well plate of the oscillation module 32 and places it at the P6 position. The first manipulator 371 Grab the deep-well plate at the P6 position and place it on the magnetic stand Mag at the P9 position, and let it stand for 1 minute; the first pipette 381 inserts the 96BC230 gun head at the TL2 position, absorbs all the supernatant in the deep-well plate on the magnetic stand at the P9 position, and transfers it to the deep-well plate at the P8 position, which is Wash1+; the first pipette 381 puts the 96BC230 gun head back to the TL2 position, and the first manipulator 371 grabs the positive screen magnetic bead deep-well plate on the magnetic stand Mag at the P9 position and places it at the P6 position; at this point, the first positive screen cleaning is completed;

[0116] Second positive screen cleaning: The first pipette 381 inserts the 96BC230 tip at the TL3 position, absorbs 200 μL of DPBS-T washing solution at the P12 position and transfers it to the positive screen magnetic bead deep-well plate at the P6 position. The first pipette 381 puts the 96BC230 tip back to the TL3 position; the first manipulator 371 grabs the deep-well plate at the P6 position and places it in the oscillation module 32, oscillating it at 1000 rpm for 1 min; the first manipulator 371 grabs the deep-well plate of the oscillation module 32 and places it at the P6 position. Take the deep-well plate at position P6 and place it on the magnetic stand Mag at position P9, and let it stand for magnetic attraction for 1 minute; the first pipette 381 inserts the 96BC230 gun head at position TL3, absorbs all the supernatant in the deep-well plate on the magnetic stand at position P9, and transfers it to the deep-well plate at position P11, which is Wash2+; the first pipette 381 puts the 96BC230 gun head back to position TL3, and the first manipulator 371 grabs the positive screen magnetic bead deep-well plate on the magnetic stand Mag at position P9 and places it at position P6; at this point, the second positive screen cleaning is completed;

[0117] The third and fourth positive screen cleaning: the first pipette 381 inserts the 96BC230 tip at the TL3 position, and repeats the second positive screen cleaning operation twice. The difference is that the supernatant magnetically sucked after the positive screen cleaning is all transferred to the first cleaning station 31, so as to complete the second cleaning of the positive screen; the first pipette 381 puts the 96BC230 tip back to the TL3 position. So far, a total of 4 positive screen cleanings have been completed;

[0118] The first pipette 381 inserts the 96BC230 tip at the TL7 position, and transfers 100 μL of H2O in the deep-well plate at the P14 position to the positive screening magnetic bead deep-well plate at the P6 position. The first manipulator 371 grabs the positive screening magnetic bead deep-well plate at the P6 position and places it in the oscillation module 32, and oscillates it at 1000 rpm for 1 min. The first manipulator 371 grabs the deep-well plate of the oscillation module 32 and places it at the P6 position. The first pipette 381 transfers 100 μL of mixed magnetic beads in the deep-well plate at the P6 position to the deep-well plate at the P10 position. The first pipette again transfers 100 μL of H2O in the deep-well plate at the P14 position. H2O is transferred to the positive screening magnetic bead deep well plate at the P6 position, the first manipulator 371 grabs the positive screening magnetic bead deep well plate at the P6 position and places it in the oscillation module 32, and oscillates it at 1000rpm for 1 minute; the first manipulator 371 grabs the deep well plate of the oscillation module 32 and places it at the P6 position, the first pipette 381 absorbs 100μL of mixed magnetic beads in the deep well plate at the P6 position and transfers it to the deep well plate at the P10 position; the first pipette 381 puts the 96BC230 gun tip back to the TL7 position; the first manipulator 371 grabs the deep well plate at the P6 position and places it at the P3 position.

[0119] Example 10

[0120] Automated positive screen product elution, reference Figure 2 , Figure 3 , Figure 4 , Fig.13 The grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid processing workstation 3 through the first transport track 2 and the first manipulator 371; the positive screening magnetic bead sample is placed at the P10 position, which is consistent with the P10 position in Example 8; the magnetic rack Mag is placed at the P9 position.

[0121] The specific operation steps are as follows: the deep well plate at position P10 is placed in the film sealing machine 61 for film sealing through the first manipulator 371, the second transport track 4, and the first grabbing structure 5; after film sealing, the positive screening magnetic bead sample deep well plate is transferred to the constant temperature incubator 36 through the first grabbing structure 5, the second transport track 4, and the first manipulator 371, and heated at 100°C for 10 minutes; after heating and eluting, the deep well plate at position P10 is placed in the film tearing machine 62 for film tearing through the first manipulator 371, the second transport track 4, and the first grabbing structure 5; after film tearing, the positive screening magnetic bead sample deep well plate is transferred to the P10 position through the first grabbing structure 5, the second transport track 4, and the first manipulator 371.

[0122] Embodiment 11

[0123] Automated positive screen elution product collection, reference Figure 2 , Figure 3 , Figure 4 , Fig.14, the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place the BC230 gun head at the TL6 position; place the deep well plate for collecting positive screening elution samples at the P4 position; place the positive screening magnetic bead sample at the P10 position, which is consistent with the P10 position in Example 9; place the magnetic stand Mag at the P9 position;

[0124] Specific operation steps: the first manipulator 371 grabs the positive screening magnetic bead sample deep well plate at the P10 position and transfers it to the magnetic stand Mag at the P9 position, and allows it to stand for magnetic attraction for 2 minutes; the first pipette 381 inserts the 96BC230 gun tip at the TL6 position, absorbs all the positive screening sample supernatant in the deep well plate on the magnetic stand at the P9 position, and transfers it to the deep well plate at the P4 position, which is the positive screening elution sample Elution+; the first pipette 381 puts the 96BC230 gun tip back to the TL6 position; the first manipulator 371 grabs the positive screening magnetic bead deep well plate on the magnetic stand Mag at the P9 position and places it at the P10 position.

[0125] Example 12

[0126] Automated qPCR detection of library enrichment, reference Figure 2 , Figure 3 , Figure 4 , Fig.15 , the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place BC90 gun tips at positions TL5 and TL7 respectively; place Wash1+ sample deep well plate at position P2, which is consistent with position P8 in Example 8; place Wash2+ sample deep well plate at position P3, which is consistent with position P11 in Example 8; place Elution+ sample deep well plate at position P14, which is consistent with position P4 in Example 10; place Elution- sample deep well plate at position P13, which is consistent with position P4 in Example 5; place 384qPCR1 plate and 384qPCR2 plate for qPCR at positions P7 and P10 respectively; place a deep well plate containing 180μL qPCR Mix in each well at position 36 of the constant temperature incubator; place a magnetic stand Mag at position P9;

[0127] The specific operation steps are as follows: insert the 96BC90 tip at the TL5 position of the first pipette 381, aspirate 18μL of qPCR Mix at the constant temperature incubator 36 position, and transfer it to the 1st row and 1st column of the 384qPCR1 plate at the P7 position; aspirate 18μL of qPCR Mix at the constant temperature incubator 36 position, and transfer it to the 1st row and 2nd column of the 384qPCR1 plate at the P7 position; aspirate 18μL of qPCR Mix at the constant temperature incubator 36 position, and transfer it to the 2nd row and 1st column of the 384qPCR1 plate at the P7 position; aspirate 18μL of qPCR Mix at the constant temperature incubator 36 position, and transfer it to the 2nd row and 2nd column of the 384qPCR1 plate at the P7 position; thus, the transfer of 384 wells of the 384qPCR1 plate is completed. Repeat the above transfer operation to complete the transfer of 384 wells of the 384qPCR2 plate;

[0128] The first pipette 381 aspirates Wash1+2 μL at position P2 and transfers it to row 1, column 1 of 384qPCR1 plate at position P7. The first pipette 381 aspirates Wash1+2 μL at position P2 and transfers it to row 1, column 1 of 384qPCR2 plate at position P10. The first pipette 381 moves to the first cleaning station 31 to clean the 96BC90 tip;

[0129] The first pipette 381 aspirates Wash2+2 μL at the P3 position and transfers it to the 1st row and 2nd column of the 384qPCR1 plate at the P7 position. The first pipette 381 aspirates Wash2+2 μL at the P3 position and transfers it to the 1st row and 2nd column of the 384qPCR2 plate at the P10 position. The first pipette 381 moves to the first cleaning station 31 to clean the 96BC90 tip.

[0130] The first pipette 381 aspirates Elution+2μL at position P14 and transfers it to the 2nd row and 1st column of the 384qPCR1 plate at position P7. The first pipette 381 aspirates Elution+2μL at position P14 and transfers it to the 2nd row and 1st column of the 384qPCR2 plate at position P10. The first pipette 381 puts the 96BC90 tip back to the TL5 position.

[0131] The first pipette 381 inserts the 96BC90 tip at the TL7 position, aspirates the Elution-2 μL at the P13 position, and transfers it to the 2nd row and 2nd column of the 384qPCR1 plate at the P7 position. The first pipette 381 aspirates the Elution-2 μL at the P13 position, and transfers it to the 2nd row and 2nd column of the 384qPCR2 plate at the P10 position. The first pipette 381 puts the 96BC90 tip back to the TL7 position.

[0132] The first manipulator 371 grabs the 384qPCR1 plate at the P7 position and places it at the P24 position, and transmits it to the grabbing range of the first grabbing structure 5 through the second transport track 4; the first grabbing structure 5 grabs the 384qPCR1 plate and places it in the film sealing machine 61 for film sealing; after the film sealing is completed, the 384qPCR1 plate is transferred to the fluorescent quantitative PCR instrument 642 through the first grabbing structure 5; according to the system program settings: 95℃3min pre-denaturation, 95℃30s denaturation, 60℃30s annealing, 72℃30s extension, 30 cycles, qPCR detection;

[0133] The first manipulator 371 grabs the 384qPCR2 plate at the P10 position and places it at the P24 position, and transmits it to the grabbing range of the first grabbing structure 5 through the second transport track 4. The first grabbing structure 5 grabs the 384qPCR2 plate and places it in the sealing machine 61 for sealing. After sealing, the 384qPCR2 plate is taken to other fluorescent quantitative PCR instruments for detection.

[0134] Embodiment 13

[0135] Automated PCR amplification, reference Figure 2 , Figure 3 , Figure 4 , Fig.16 , the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; place BC230 gun tips at the TL6 position respectively; place PCR1, PCR2, PCR3, PCR4, PCR5, and PCR6 plates at the P3, P4, P6, P10, P12, and P14 positions respectively; place a deep well plate containing 500 μL PCR Mix in each well at the P8 position; place Elution+ at the constant temperature incubator 36 position, which is consistent with the Elution+ sample at the P14 position in Example 11; place the magnetic stand Mag at the P9 position;

[0136] The specific operation steps are as follows: the first pipette 381 inserts the 96BC230 tip at the TL6 position, absorbs 100 μL of Elution+ at the constant temperature incubator 36 position, and transfers it to the PCR Mix deep well plate at the P8 position, and the first pipette 381 aspirates up and down to mix; the first pipette 381 absorbs 100 μL of PCR Mix at the P8 position, and transfers it to the PCR1 plate at the P3 position; the first pipette 381 absorbs 100 μL of PCR Mix at the P8 position, and transfers it to the PCR2 plate at the P4 position; the first pipette 381 absorbs 100 μL of PCR Mix at the P8 position, and transfers it to the PCR3 plate at the P6 position; the first pipette 381 absorbs 100 μL of PCR Mix at the P8 position, and transfers it to the PCR4 plate at the P10 position; the first pipette 381 absorbs 100 μL of PCR Mix at the P8 position, and transfers it to the PCR5 plate at the P12 position; the first pipette 381 absorbs 100 μL of PCR Mix at the P8 position 100 μL, transferred to PCR6 plate at position P14;

[0137] Then these PCR plates were transferred to a sealing machine for sealing. After sealing, PCR amplification was performed. The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The number of cycles was based on the qPCR results.

[0138] Embodiment 14

[0139] Automated SA magnetic bead cleaning, reference Figure 2 , Figure 3 , Figure 4 , Fig.17 , the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; BC230 gun tips are placed at positions TL3 and TL6, a reagent tank containing 20mL 4M NaCl is placed at position P7, a reagent tank containing 110mL DPBS-T washing solution is placed at position P10, an empty deep-well plate for washing solution is placed at position P11, a deep-well plate containing 50μL SA magnetic beads is placed at position 36 of the constant temperature incubator, and a magnetic stand Mag is placed at position P9;

[0140] The specific operation steps are as follows: the first pipette 381 is inserted with the 96BC230 tip at the TL6 position, 150 μL of the DPBS-T washing solution at the P10 position is sucked, and transferred to the deep-well plate at the P11 position, and the above DPBS-T washing solution transfer operation is repeated 6 times;

[0141] The first pipette 381 absorbs 150 μL of DPBS-T washing solution at the P11 position, and transfers it to the SA magnetic bead deep-well plate at the constant temperature incubator 36 position. The first manipulator 371 grabs the deep-well plate at the constant temperature incubator 36 position and places it in the oscillation module 32, and oscillates it at 1000 rpm for 1 minute. The first manipulator 371 grabs the deep-well plate of the oscillation module 32 and places it at the P6 position. The first manipulator 371 grabs the deep-well plate at the P6 position and places it on the magnetic stand Mag at the P9 position, and allows it to stand for 2 minutes. The first pipette 381 absorbs all the supernatant in the deep-well plate at the P9 position, and transfers it to the first cleaning station 31, and cleans the tip of the first pipette 381 here. The first manipulator 371 grabs the deep-well plate at the P9 position and places it in the constant temperature incubator 36 position. At this point, one SA magnetic bead cleaning is completed.

[0142] Repeat the above experimental operation twice again to complete the three washings of SA magnetic beads; then, the first pipette 381 puts the 96BC230 tip back to the TL6 position; the first pipette 381 inserts the 96BC230 tip at the TL3 position, draws 120 μL of 4MNaCl at the P7 position, and transfers it to the SA magnetic bead deep well plate at the constant temperature incubator 36 position; the first pipette 381 puts the 96BC230 tip back to the TL3 position.

[0143] Embodiment 15

[0144] Automated PCR product incubation with SA magnetic beads, reference Figure 2 , Figure 3 , Figure 4 , Fig.18 , the grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; the BC230 gun head is placed at the TL3 position, which is consistent with the position in Example 13; the deep well plate containing SA magnetic beads and NaCl is placed at the P8 position, which is consistent with the sample at the constant temperature incubator 36 position in Example 13; the PCR1, PCR2, PCR3, PCR4, PCR5, and PCR6 plates after PCR amplification are placed at the P3, P4, P6, P10, P12, and P14 positions respectively; the magnetic stand Mag is placed at the P9 position;

[0145] The specific operation steps are as follows: the first pipette 381 inserts the 96BC230 gun tip at the TL3 position, and successively aspirates 100 μL of the samples in the PCR1, PCR2, PCR3, PCR4, PCR5, and PCR6 plates at the P3, P4, P6, P10, P12, and P14 positions, and sequentially transfers them to the SA magnetic beads and NaCl deep-well plate at the P8 position; the first pipette 381 puts the 96BC230 gun tip back to the TL3 position;

[0146] Sealing: The first manipulator 371 grabs the deep-well plate at position P8 and places it at position P24, and transmits it to the grabbing range of the first grabbing structure 5 through the second transport track 4; the first grabbing structure 5 grabs the deep-well plate and places it in the sealing machine 61 for sealing; after sealing, the deep-well plate is transferred to the oscillation module 32 through the first grabbing structure 5, the second transport track 4 and the first manipulator 371, and the PCR product and SA magnetic beads are fully oscillated and incubated for 20 minutes at 1000rpm;

[0147] Film tearing: After the incubation is completed, the first manipulator 371 grabs the deep-well plate of the oscillation module 32 and places it at the P24 position, and transmits it to the grabbing range of the first grabbing structure 5 through the second transport track 4; the first grabbing structure 5 grabs the deep-well plate and places it in the film sealing machine 61 for film tearing; after the film tearing is completed, the deep-well plate is transferred to the P8 position through the first grabbing structure 5, the second transport track 4 and the first manipulator 371.

[0148] Example 16

[0149] Automated SA magnetic bead single-strand preparation, reference Figure 2 , Figure 3 , Figure 4 , Fig.19 The grippers in the consumables stack 1 grab the required reagent consumables respectively, and transport the required reagent consumables to the table in the liquid handling workstation 3 through the first transport track 2 and the first manipulator 371; BC230 gun tips are placed at positions TL2, TL6, TL7, and TL8, BC90 gun tips are placed at position TL3, a deep well plate containing 10 μL 1M HCl per well is placed at position P3, a reagent tank containing 20mL 40mM NaOH is placed at position P12, and a reagent tank containing 20mL The reagent tank of 2×DPBS, the empty deep-well plate for 2×DPBS is placed at position P15; the sample after incubation of PCR product and SA magnetic beads is placed at position P8, which is consistent with the position P8 in Example 14; the DPBS-T washing solution is placed at position P14, which is consistent with the sample at position P11 in Example 13; the empty deep-well plate for collecting ssDNA samples is placed at position P4, which is the ssDNA screened in this round; the empty deep-well plate for collecting the supernatant after incubation of PCR product and SA magnetic beads is placed at position P10; the magnetic stand Mag is placed at position P9;

[0150] The specific operation steps are as follows: the first pipette 381 inserts the 96BC230 tip at the TL2 position, absorbs 200 μL of 2×DPBS at the P12 position, and transfers it to the deep-well plate at the P15 position; the first pipette 381 puts the 96BC230 tip back to the TL2 position;

[0151] The first manipulator 371 grabs the PCR product and SA magnetic beads incubated sample deep-well plate at position P8 and transfers it to the magnetic stand Mag at position P9, and allows it to stand for 2 minutes; the first pipette 381 inserts the 96BC230 gun tip at position TL6, absorbs all sample supernatants in the deep-well plate on the magnetic stand at position P9, and transfers them to the deep-well plate at position P10; the first pipette 381 puts the 96BC230 gun tip back to position TL6; the first manipulator 371 grabs the deep-well plate on the magnetic stand Mag at position P9 and places it at position P6;

[0152] First SA magnetic bead washing: The first pipette 381 inserts the 96BC230 tip at the TL7 position, absorbs 200 μL of DPBS-T washing solution at the P14 position and transfers it to the SA magnetic bead deep-well plate at the P6 position. The first pipette 381 puts the 96BC230 tip back to the TL7 position; the first manipulator 371 grabs the deep-well plate at the P6 position and places it on the oscillation module 32, oscillating it at 1000 rpm for 1 min; the first manipulator 371 grabs the deep-well plate at the oscillation module 32 and places it on the P6 position. The hand 371 grabs the deep-well plate at the P6 position and places it on the magnetic stand Mag at the P9 position, and leaves it to stand for magnetic attraction for 1 minute; the first pipette 381 inserts the 96BC230 gun tip at the TL7 position, absorbs all the supernatant in the deep-well plate on the magnetic stand at the P9 position, and transfers it to the first cleaning station 31; the first pipette 381 puts the 96BC230 gun tip back to the TL7 position, and the first manipulator 371 grabs the SA magnetic bead deep-well plate on the magnetic stand Mag at the P9 position and places it at the P6 position; at this point, the first SA magnetic bead cleaning is completed;

[0153] Second, third, and fourth SA cleaning: Repeat the above cleaning operation 3 times to complete the 3 cleanings of SA magnetic beads. So far, a total of 4 SA magnetic beads cleanings have been completed;

[0154] Alkaline elution: The first pipette 381 inserts the 96BC230 tip at the TL8 position, absorbs 100 μL of 40 mM NaOH on the constant temperature incubator 36 position and transfers it to the SA magnetic bead deep well plate at the P6 position, and the first manipulator 371 grabs the SA magnetic bead deep well plate at the P6 position and places it on the oscillation module 32, and oscillates it at 1000 rpm for 2 minutes; the first manipulator 371 grabs the deep well plate of the oscillation module 32 and places it at the P6 position, and the first manipulator 371 grabs the deep well plate at the P6 position and places it on the magnetic stand Mag at the P9 position, and allows it to stand for magnetic attraction for 2 minutes; the first pipette 381 absorbs 100 μL of elution supernatant from the deep well plate on the magnetic stand at the P9 position, and transfers it to the deep well plate at the P4 position for collecting ssDNA; the first pipette 381 puts the 96BC230 tip back to the TL8 position, and the first manipulator 371 grabs the SA magnetic bead deep well plate on the magnetic stand Mag at the P9 position and places it at the P8 position;

[0155] The first pipette 381 picks up the 96BC90 pipette tips at the TL3 position, aspirates 3.75 μL of 1 M HCl at the P3 position and transfers it to the ssDNA deep well plate at the P4 position. Then the first pipette 381 returns the 96BC90 pipette tip to the TL3 position. The first pipette 381 picks up the 96BC230 pipette tip at the TL2 position, aspirates 100 μL of 2×DPBS at the P15 position and transfers it to the ssDNA deep well plate at the P4 position. Then the first pipette 381 returns the 96BC230 pipette tip to the TL2 position.至此,单链DNA制备完毕。

[0156] Data analysis:

[0157] By adopting the technical solution of the present application, one round of automated screening of aptamers can be completed, including reverse screening and forward screening;

[0158] As shown in Example 12, whether the screening of aptamers is completed or not depends on the enrichment of the library in each round of screening, that is, observing the qPCR results of the forward and reverse screening of the library corresponding to the target in each round of screening to determine the enrichment of the library. As the number of screening rounds increases, the Cq value of the forward screening of the library corresponding to the target continuously decreases and tends to be stable, the Cq value of the reverse screening continuously increases and tends to be stable, and the Cq value of the forward screening of the library corresponding to the target is earlier than the Cq value of the reverse screening (i.e., Cq value: Elution+ < Elution-, hereinafter referred to as E+ < E-). At this time, the screening can be stopped.

[0159] 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 data analysis drawings of the present invention. Obviously, what is described here is only a part of the examples of the present invention, not all of the examples. All other examples obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the protection scope of the present invention.

[0160] The present invention only shows the analysis data of a certain batch of 90-target screening, including but not limited to the data of other batches.

[0161] Based on the target immobilized magnetic bead method screening, using the above-mentioned aptamer automated screening method, a total of 5 rounds of screening were carried out, named R1, R2, R3, R4, and R5 rounds respectively. The screening situation of each round is shown in Table 1:

[0162] Table 1 Screening situation of different rounds of 90-target aptamers:

[0163]

[0164] As shown in Example 12, the positive selection elution products (E+) and negative selection elution products (E-) of 90 targets in each round will undergo qPCR verification. Correspondingly, each target will generate Cq values for the corresponding elution products (E+ and E-); therefore, we conducted statistical analyses as follows Fig. 20 and Fig.21 : The Cq values of the positive selection elution products and negative selection elution products corresponding to 90 targets in each round were subjected to violin statistical analysis; the results showed that as the number of screening rounds increased, the positive selection Cq values of the libraries corresponding to the targets continuously decreased and tended to be stable, while the negative selection Cq values continuously increased and tended to be stable, indicating that the screening libraries were continuously enriched;

[0165] As described above, we also statistically analyzed the qPCR conditions of the positive and negative selection elution products of 90 targets in each round. Specifically, the following statistics were performed on the Cq values of the positive and negative selection elution products corresponding to each target in each round; as shown in Table 2, combined with Fig. 20 and Fig.21 , as the number of screening rounds increased, the positive selection Cq values of the libraries corresponding to the targets continuously decreased and tended to be stable, the negative selection Cq values continuously increased and tended to be stable, and the positive selection Cq values of the libraries corresponding to the targets were earlier than the negative selection Cq values (E+ < E-); among them, there were only 5 cases where the positive selection Cq values were earlier than the negative selection Cq values in the third round. This was due to the expansion and high complexity of the library before the third round of screening. After the third round of screening, the library gradually became enriched; however, generally speaking, as the screening rounds progressed, the positive selection Cq values of the libraries corresponding to the targets were earlier than the negative selection Cq values;

[0166] Table 2:

[0167]

[0168] As shown in Example 16, the single-stranded products (ssDNA) prepared from 90 targets in each round will undergo fragment analyzer analysis and verification to ensure that the band sizes of the single-stranded products prepared from each target in each round are correct and the peaks are single; as Fig. 22 and Fig.23 shown, the verification results of the single-stranded products in a certain round during the 90-target screening process are specifically presented. The results indicate that the band sizes of the single-stranded products corresponding to each target are correct and the peaks are single, and the quality of the single-stranded DNA is fine;

[0169] Surface plasmon resonance (SPR) analysis was used to identify the nucleic acid aptamers obtained after screening: After the obtained enriched library products were analyzed by high-throughput sequencing, several sequences were selected for each target and synthesized by Genewiz (Jiangsu) Biotechnology Co., Ltd., and their affinities were detected. In subsequent tests, for some targets, 1 sequence with the strongest binding ability was determined from the 12 sequences synthesized and selected in the final 5th round, and the affinities were all at the nanomolar level; as Fig.24As shown, 37 out of 90 targets measured by SPR were successfully screened for nucleic acid aptamers, with a success rate of 41%.

[0170] The technical solution of the present invention is described above in conjunction with specific implementation methods, but it should be noted that the above descriptions are only for explaining the solution of the present invention and cannot be interpreted in any way as a specific limitation on the scope of protection of the invention. Based on the explanation here, those skilled in the art can think of other specific implementation methods or equivalent replacements of the present invention without creative work, and they will all fall within the scope of protection of the present invention.

Claims

1. A method for automated screening of nucleic acid aptamers, characterized in that: The method comprises: S1: Preparation of reagents, consumables and automated workstations. Before automated screening, reagents, consumables and instruments must be prepared. The automated workstation includes instruments and equipment, and the preparation of the automated workstation includes initialization of instruments and equipment. S2: library denaturation and renaturation treatment, the single-stranded DNA random library is denatured and renatured by an automated workstation; S3: Counter-screening magnetic bead washing: automatic counter-screening magnetic bead washing is performed through an automated workstation, the washing solution is aspirated into a counter-screening magnetic bead deep-well plate, and after oscillation, the supernatant is aspirated and transferred to the washing station to complete the counter-screening magnetic bead washing; S4: Incubation of the reverse screening magnetic beads and the library. The reverse screening magnetic beads and the library are incubated automatically in the automated workstation. The renatured library is transferred to the reverse screening magnetic bead deep-well plate. After processing, the reverse screening magnetic beads and the library are incubated. S5: Back-screen cleaning, automatically control the automated workstation to perform automated back-screen cleaning; S6: collecting the back-screening elution product, collecting the back-screening elution product through an automated workstation; S7: Incubate the supernatant with the protein label. Use an automated workstation to seal the deep-well plate containing the supernatant and the protein label, and then fully shake and incubate. S8: Positive screening magnetic bead washing and incubation, automatic control of the automated workstation for automated positive screening magnetic bead washing and positive screening magnetic bead incubation with the library; S9: Main screen cleaning, automatic main screen cleaning is performed through an automated workstation; S11: positive screen elution product collection, automatic positive screen elution product collection is performed through an automated workstation; S12: qPCR detection of library enrichment, qPCR detection is performed by an automated workstation; S13: PCR amplification: automatic PCR amplification is performed by an automated workstation, the positive screening elution product is transferred to the PCR Mix plate for PCR amplification; S14: SA magnetic bead washing: automatic SA magnetic bead washing is performed through an automated workstation, the washing solution is aspirated into the SA magnetic bead deep-well plate, and the supernatant in the SA magnetic bead deep-well plate is aspirated after treatment, and transferred to the washing station to complete the SA magnetic bead washing; S15: Incubation of PCR products with SA magnetic beads is performed by an automated workstation to complete the incubation of PCR products with SA magnetic beads; S16: SA magnetic bead single-strand preparation, which is completed by operating on an automated workstation.

2. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: In the step S2, when the single-stranded DNA random library is subjected to denaturation and renaturation treatment by the automated workstation, in the automated workstation, the DPBS buffer is first aspirated and transferred to the well plate containing the random library, and then placed in an oscillation module for sufficient oscillation. After sufficient oscillation, the single-stranded DNA random library is aspirated and transferred to the PCR plate, and the film is sealed. After the film is sealed, the single-stranded DNA random library is transferred to the PCR instrument for denaturation and renaturation treatment.

3. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: In the step S5, automated back-screening cleaning is performed by automatically controlling an automated workstation. In the automated workstation, the back-screening sample deep-well plate is transferred to the magnetic stand Mag and allowed to stand for magnetic attraction. The back-screening sample supernatant is transferred to the deep-well plate containing the protein label, and then the DPBS-T washing solution is transferred to the back-screening magnetic bead deep-well plate. After sufficient oscillation, the plate is allowed to stand for magnetic attraction. The supernatant is transferred to the cleaning station to complete the back-screening cleaning.

4. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: In the step S6, the back screening elution product is collected by an automated workstation, and in the automated workstation, the eluted back screening magnetic bead sample deep well plate is transferred to a magnetic stand Mag for static magnetic absorption, and the back screening sample supernatant is sucked and transferred to the deep well plate, which is recorded as the back screening elution sample Elution-; In the step S11, the positive screen elution product is automatically collected by an automated workstation, the eluted positive screen magnetic bead sample deep well plate is transferred to the magnetic stand Mag for static magnetic absorption, and the positive screen sample supernatant is transferred to the deep well plate, which is recorded as the positive screen elution sample Elution+.

5. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: In the step S8, the automatic positive screening magnetic beads are automatically cleaned and the positive screening magnetic beads are incubated with the library by automatically controlling the automated workstation. In the automated workstation, DPBS-T washing solution is added to the positive screening magnetic beads deep well plate, and then the plate is transferred to the oscillation module for sufficient oscillation, and then the plate is transferred to the magnetic stand Mag for static magnetic absorption, the supernatant of the positive screening magnetic beads deep well plate is transferred to the cleaning station, and the positive screening magnetic beads deep well plate is transferred to the constant temperature incubator to complete the positive screening magnetic beads cleaning; subsequently, the sample containing the reverse screening supernatant and the protein label is transferred to the positive screening magnetic beads deep well plate, and the plate is fully oscillated and incubated in the oscillation module to complete the incubation of the positive screening magnetic beads with the library.

6. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: In the step S9, automated positive screen cleaning is performed by an automated workstation. In the automated workstation, DPBS-T washing solution is added to the positive screen magnetic bead deep-well plate, which is transferred to an oscillation module for sufficient oscillation. After magnetic attraction, the supernatant is aspirated and transferred to the deep-well plate, which is recorded as Wash1+, to complete the first positive screen cleaning. The above steps are repeated, and after magnetic attraction, the supernatant is aspirated and transferred to another deep-well plate, which is recorded as Wash2+, to complete the second positive screen cleaning.

7. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: In the step S12, an automated workstation is used to transfer the qPCR Mix in the constant temperature incubator to the 384 wells of the 384qPCR1 plate, and Wash1+, Wash2+, Elution+, and Elution- are respectively aspirated and transferred to the 384qPCR1 plate. The 384qPCR1 plate is placed in a film sealing machine and sealed, and then transferred for qPCR detection; the above steps are repeated to complete the parallel qPCR detection of the 384qPCR2 plate.

8. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: In the step S15, the operation is performed through an automated workstation. In the automated workstation, the samples in the PCR1 plate to the PCR6 plate are first aspirated and transferred to deep-well plates containing SA magnetic beads and NaCl, respectively, and then sealed. After sealing, the plates are transferred to an oscillation module, and the PCR products and the SA magnetic beads are fully oscillated and incubated.

9. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: In the step S16, the operation is performed through an automated workstation. In the automated workstation, the SA magnetic beads are first cleaned after incubation, and then alkaline elution is performed. The eluted supernatant is transferred to a deep-well plate for collecting ssDNA products, and HCl and 2×DPBS are aspirated and transferred to the deep-well plate for ssDNA products to complete the single-stranded preparation.

10. The method for automated screening of nucleic acid aptamers according to claim 1, characterized in that: The automated workstation comprises a frame, on which a consumables stacking area (1), a liquid processing workstation (3), a first transverse transport track (2) for transporting consumables, a second longitudinal transport track (4) for transporting consumables, and a first gripping structure (5) are provided, wherein the consumables stacking area (1) is arranged on one side of the liquid processing workstation (3), and the first gripping structure (5) is located at the rear of the liquid processing workstation (3); The liquid handling workstation (3) comprises a first cleaning station (31), a vortex oscillation module (32), a PCR module (33), a second cleaning station (34), a garbage station (35), a constant temperature incubator (36), a second gripping structure (37) and a pipette (38), wherein the second gripping structure (37) comprises a first manipulator (371) and a second manipulator (372); The frame is also provided with a film sealing machine (61), a film tearing machine (62), a centrifuge (63), a fragment analyzer (641), a fluorescence quantitative PCR instrument (642), and an enzyme marker (643); the film sealing machine (61), the film tearing machine (62), and the centrifuge (63) are all arranged on the right side of the first grasping structure (5); and the fragment analyzer (641), the fluorescence quantitative PCR instrument (642), and the enzyme marker (643) are arranged on the left side of the first grasping structure (5).

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