WO2024065646A1 - 生物芯片的自动化控制方法及其自动化控制系统 - Google Patents

生物芯片的自动化控制方法及其自动化控制系统 Download PDF

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Publication number
WO2024065646A1
WO2024065646A1 PCT/CN2022/123191 CN2022123191W WO2024065646A1 WO 2024065646 A1 WO2024065646 A1 WO 2024065646A1 CN 2022123191 W CN2022123191 W CN 2022123191W WO 2024065646 A1 WO2024065646 A1 WO 2024065646A1
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biochip
reagent
biochips
waste liquid
permeabilization
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PCT/CN2022/123191
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English (en)
French (fr)
Inventor
何福涛
雷晓娟
陈安
丘志丰
洪艳
万志远
沈梦哲
陈奥
黎宇翔
章文蔚
徐讯
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深圳华大生命科学研究院
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Priority to PCT/CN2022/123191 priority Critical patent/WO2024065646A1/zh
Publication of WO2024065646A1 publication Critical patent/WO2024065646A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present disclosure relates to the field of automatic operation of biochips, and in particular to an automatic control method of a biochip and an automatic control system thereof.
  • Spatial Transcriptomics measures the total mRNA of a complete tissue section, combines the spatial information of the total mRNA with the morphological content, and plots the locations where all gene expressions occur, obtaining a complex and complete gene expression map of the biological process.
  • Microscopic imaging and sequencing technology can be combined to obtain gene expression data while retaining the spatial location information of the sample to the greatest extent, providing important information on the relationship between cell function, phenotype, and location in the tissue microenvironment.
  • Spatial transcriptomics technology obtains spatial location and corresponding gene expression information. By analyzing spatial transcriptomics data, we can know which signal transduction pathways are activated in certain cells. scientistss can select genes of interest through the data generated by spatial transcriptomics technology and display their spatially discernible expression on the original tissue sections. Since all mRNAs are captured, they are no longer limited to viewing only a single gene, but can select any number of genes in any combination to view and analyze together. These research processes require biochemical reactions to convert information in biological tissues into a detectable form, which involves multiple reagents and usually requires cumbersome manual operations to repeat multiple rounds or steps of biochemical reactions with different principles.
  • 10X Genomics mainly provides solutions based on manual operation, and the supporting instrument solutions include: Nano String's laser capture microdissection (Laser capture dissection) based on Leica's BondRX immunohistochemical stainer and GeoMx platform for multiple staining and imaging; Akoya's multiple staining and in situ imaging based on PhenoCycler-Fusion.
  • ROIs target regions of interest
  • the present disclosure provides an automated control method for a biochip, including the operations of sucking up a reagent by a pipetting mechanism carrying a suction component, and adding the reagent to the biochip by a pipetting mechanism carrying the suction component that has sucked up the reagent.
  • the operations include at least one of the following steps:
  • Incubation The incubation reagent is sucked by the suction component carried by the pipetting mechanism, and the incubation reagent is added to the biochip by the suction component carried by the pipetting mechanism that has sucked the incubation reagent;
  • Tissue permeabilization a pipetting mechanism carries a suction component to absorb a permeabilization reagent, and a pipetting mechanism carries a suction component that has absorbed the permeabilization reagent to add the permeabilization reagent to the biochip;
  • Reverse transcription the reverse transcription reagent is sucked by the suction component carried by the pipetting mechanism, and the reverse transcription reagent is added to the biochip by the suction component carried by the pipetting mechanism that has sucked the reverse transcription reagent;
  • Tissue removal The pipetting mechanism carries the suction component to absorb the tissue removal reagent, and the pipetting mechanism carries the suction component that has absorbed the tissue removal reagent to add the tissue removal reagent to the biochip.
  • waste liquid is pumped away by a waste liquid pumping mechanism.
  • tissue removal step at least one of the following steps is further included:
  • Nucleic acid release The nucleic acid release reagent is sucked by the suction component carried by the pipetting mechanism, and the nucleic acid release reagent is added to the biochip by the suction component carried by the pipetting mechanism that has sucked the nucleic acid release reagent, thereby completing the nucleic acid release;
  • the released nucleic acid is sucked up by a pipetting mechanism carrying a suction component, and the released nucleic acid is stored.
  • a washing step is performed between the incubation step and the tissue permeabilization step and/or a washing step is performed between the tissue permeabilization step and the reverse transcription step and/or a washing step is performed between the reverse transcription step and the tissue removal step and/or a washing step is performed between the tissue removal step and the nucleic acid release step.
  • the cleaning step is: using a pipetting mechanism to carry a suction component to absorb the cleaning reagent, using a pipetting mechanism to carry the suction component that has absorbed the cleaning reagent to add the cleaning reagent to the biochip, and using a waste liquid extraction mechanism to extract the cleaning waste liquid.
  • steps of the operation are performed automatically.
  • the currently executed step and/or the current status is displayed to the user in real time.
  • a plurality of biochips are arranged in a biochip box, information of the plurality of biochips is associated with information of the biochip box by scanning, and permeabilization times of the plurality of biochips are input into the system.
  • a biochip carrying a target tissue is provided, the size of which is customized.
  • a plurality of biochips are sequentially arranged in at least one row in a biochip box.
  • the permeabilization reagent waste liquid is simultaneously extracted for the biochips in the row ⁇ the cleaning reagent is added ⁇ the cleaning reagent waste liquid is extracted ⁇ the reverse transcription reagent is added;
  • the waste liquid of the permeabilization reagent is first extracted from the biochip in the first row and the cleaning reagent is added, then the waste liquid of the permeabilization reagent is extracted from the biochip in the second row and the cleaning reagent is added, then the waste liquid of the cleaning reagent is extracted from the biochip in the first row and the reverse transcription reagent is added, and then the waste liquid of the cleaning reagent is extracted from the biochip in the second row and the reverse transcription reagent is added.
  • the permeabilization times of the plurality of biochips are not the same, and the plurality of biochips are arranged in order of the permeabilization time from the smallest to the largest,
  • the permeabilization reagent waste liquid is extracted from the multiple biochips in the order of completion of the permeabilization time, the cleaning reagent is added, the cleaning reagent waste liquid is extracted, and the reverse transcription reagent is added;
  • the permeabilization reagent waste liquid is first extracted from each biochip in the first row in the order of completion of the permeabilization time, the cleaning reagent is added, the cleaning reagent waste liquid is extracted, and the reverse transcription reagent is added. Then, the permeabilization reagent waste liquid is extracted from each biochip in the second row in the order of completion of the permeabilization time, the cleaning reagent is added, the cleaning reagent waste liquid is extracted, and the reverse transcription reagent is added; and
  • the automated control method includes a waste liquid extraction step, and the waste liquid extraction step includes the following steps:
  • the waste liquid pumping mechanism moves to the top of the biochip box, starts the pump of the waste liquid pumping mechanism to first pump out the waste liquid at a higher position in the biochip box through the waste liquid pumping component, and then pumps out the waste liquid at a lower position in the biochip box, raises the waste liquid pumping component along the Z axis and turns off the pump, and moves the waste liquid pumping mechanism to the cleaning area for cleaning;
  • the waste liquid of the first row of biochip reactions is first sucked out, and then the waste liquid of the second row of biochip reactions is sucked out.
  • the sucking steps are the same as the sucking steps when the multiple biochips are arranged in one row.
  • the system scans the information of the biochip box and the information of the biochips in the biochip box to determine whether the position of the biochip in the biochip box, the number of biochips, and the information of the biochips match the information in the system:
  • the existing biochip cartridge is not in the reverse transcription step or the nucleic acid release step, remind the user that it is necessary to wait for a period of time X before transferring the biochip cartridge to the reaction area to perform the operation;
  • the biochip box is transferred to the reaction area by a gripping mechanism to perform the operation, and the gripping mechanism and the pipetting mechanism work independently of each other.
  • the gripping mechanism and/or the pipetting mechanism do not touch the biochip.
  • the biochip box is capped with a sealing cover and/or a temperature-controlled cover and opened with a gripping mechanism.
  • the biochip box in the product collection step, is first transferred to the inclined area by a gripping mechanism, and the product collection is completed in the inclined area.
  • the pipetting mechanism in the product collection step, carries the suction component to absorb the reagent, the pipetting mechanism carries the suction component that has absorbed the reagent to flush the biochip in the biochip box, and the pipetting mechanism carries the suction component to collect the product.
  • a step of taking a suction component by a pipetting mechanism includes: determining whether the current column in the suction component container meets the conditions for taking the suction component; when the quantity and position meet the conditions, the pipetting mechanism moves to the top of the suction component in this column, lowers the height, and takes the suction component in this column at the same time; if the current column does not meet the conditions for taking the suction component, polls the next column until the suction component container is polled, and starts polling the next suction component container until all suction component containers are polled; if there is no column that meets the conditions for taking the suction component, remind the user to replenish the suction component.
  • the step of the pipetting mechanism sucking up the reagent includes: the pipetting mechanism carries the suction component to the top of the reagent area, the height is lowered, and the reagent liquid level is detected. If the reagent is not detected, the user is reminded to replenish the reagent. If the reagent is detected, it is determined whether the surplus amount meets the amount to be sucked up by the carried suction component. If not, the user is reminded to replenish the reagent. If so, it is further determined whether the reagent to be added is a cleaning reagent. If it is a cleaning reagent, the height of all the suction components is lowered to suck up the cleaning reagent together. If it is not a cleaning reagent, the height of each suction component is lowered separately to suck up the reaction reagent.
  • the automated control methods are applied to transcriptomics, proteomics, metabolomics and/or lipidomics labeling or capture and pathology markers.
  • the present disclosure also provides an automated control system for a biochip, which includes a biochip box, a reaction area, a pipetting mechanism, and a gripping mechanism, and is configured as the automated control method for a biochip.
  • the automated control method and automated control system provided by the present disclosure can realize scalable automatic scheduling and control of multiple biochips, realize the labeling and capture of biological information in tissues, and the analysis of multiple omics such as transcriptome, proteome, and immunology, and realize the collection function of the captured products.
  • the method and system provided by the present disclosure meet the processing requirements of multiple biochips, high utilization, high throughput, and high automation.
  • FIG1 is an overall flow chart of the automated control method of a biochip disclosed herein;
  • FIG2 is a flow chart of taking a pipette tip in the automated control method of a biochip disclosed in the present invention
  • FIG3 is a flow chart of adding reagents in the automated control method of the biochip disclosed in the present invention.
  • FIG4 is a flow chart of the incubation steps of the automated control method of the biochip disclosed herein;
  • FIG5 is a flow chart of the cleaning steps of the automated control method for a biochip disclosed herein;
  • FIG6 is a flow chart of the tissue permeabilization step of the automated control method of the biochip disclosed herein;
  • FIG. 7 is a schematic diagram of two rows of biochips with different permeabilization times according to the present disclosure.
  • FIG8 is a flow chart of the reverse transcription step of the automated control method of the biochip disclosed herein;
  • FIG9 is a flow chart of the tissue removal step of the automated control method of the biochip disclosed herein;
  • FIG10 is a flow chart of the nucleic acid release step of the automated control method of the biochip disclosed herein;
  • FIG. 11 is a flow chart of the collection steps of the automated control method of the biochip disclosed herein.
  • FIG. 12 is a flow chart of the waste liquid extraction step of the automated control method of the biochip disclosed herein.
  • the present disclosure provides an automated control method for a biochip and an automated control system for implementing the automated control method.
  • the automatic control method of the present disclosure includes a pre-execution phase and an execution phase.
  • biochips are bound to the biochip box by scanning a code, and the permeabilization time for each biochip is entered, for example, by manual entry.
  • Place consumables (pipette parts such as pipette tips, reagents, etc.), for example, by manual placement.
  • the program enters the process of determining the status of the biochip box in the system. If the biochip box in the system is not in the reverse transcription or nucleic acid release stage, a pop-up window will remind the user: it is necessary to wait for time X before transferring the biochip box to the reaction area. If all the biochip boxes in the system are in the reverse transcription or nucleic acid release stage, the next determination process will be entered.
  • the grasping mechanism is controlled to clamp the biochip box from the loading area and transfer it to the reaction area.
  • the grasping mechanism and the liquid transfer mechanism work independently.
  • the grasping mechanism includes a clamping claw
  • the liquid transfer mechanism includes a multi-channel liquid transfer mechanical arm.
  • the system executes the biochemical reaction according to the selected execution process.
  • the automated control method disclosed in the present invention only requires manual input of execution information and placement of execution consumables and reagents before the execution begins. No human intervention is required during the entire execution phase.
  • the software interface can display the current execution steps and status in real time, and can display the estimated remaining execution time. Operators can predict and arrange operations in advance according to the situation, saving manpower.
  • the execution phase enables the capture and product collection of spatial omics (e.g., transcriptomics, proteomics, metabolomics, and lipidomics), which includes the following steps:
  • the step of taking a pipette tip includes: determining whether the current column meets the conditions for taking a pipette tip, and if and only if both the quantity and the position meet the conditions, the conditions are met, the pipetting mechanism moves to the top of the pipette tip in the column, the Z axis (height direction) descends, and the pipette tip is taken from the column at the same time.
  • the step of adding reagents includes: determining whether the biochips in the biochip box are in one row or two rows:
  • liquid may be added to the biochips in one row at a time.
  • the biochips are arranged in two rows, add liquid to the first row of biochips first: take the pipette tip for the first row of biochips, transfer the pipette tip with the pipette tip to the top of the reagent area, and lower the Z axis (height direction) to detect the reagent liquid level. If the reagent is not detected, a pop-up window will remind the user: Please add reagent. If it is detected, determine whether the surplus amount is enough for the amount to be absorbed by the pipette tip. If not, a pop-up window will remind the user: Please add reagent. If the surplus amount is sufficient, determine whether the reagent to be added is a cleaning reagent.
  • the reagent adding step if the biochips are arranged in three or more rows, the reagent adding step described above for the biochips arranged in only one row is sequentially performed for the three or more rows of biochips.
  • the incubation step includes: determining whether the plurality of biochips are arranged in one row or two rows in the biochip box:
  • the pipette tips are taken for this row of biochips (the process of taking the pipette tips is shown in Figure 3), and the pipetting mechanism, such as a multi-channel pipetting robot, carries the pipette tips to the reagent area, extracts a certain amount of cleaning reagent with a higher concentration (such as 5*SSC 400 ⁇ L) (see Figure 3 for the process of extracting reagents), adds the cleaning reagent to this row of biochips, and then the waste liquid extraction mechanism extracts the waste liquid of the cleaning reagent.
  • the pipetting mechanism such as a multi-channel pipetting robot, carries the pipette tips to the reagent area, extracts a certain amount of cleaning reagent with a higher concentration (such as 5*SSC 400 ⁇ L) (see Figure 3 for the process of extracting reagents), adds the cleaning reagent to this row of biochips, and then the waste liquid extraction mechanism extracts the waste liquid of the cleaning reagent.
  • the pipetting mechanism carries multiple pipette tips to the top of the reaction area, descends in the Z-axis (height direction), spits the incubation reagent to the side of the biochip, waits for a predetermined time, such as 5 minutes, and sucks and pumps the side multiple times, such as 5 times.
  • the pipetting mechanism carries multiple pipette tips to the top of the trash can and discards the pipette tips.
  • the suction component of the waste liquid extraction mechanism such as a steel needle, moves to the top of the biochip box, and the steel needle descends to extract the incubation reagent waste liquid for this row of biochips.
  • the pipette mechanism such as a multi-channel pipette robot, carries the pipette tip to the reagent area, extracts a certain amount of cleaning reagent with a higher concentration (such as 5*SSC 400 ⁇ L) (see Figure 3 for the process of extracting the reagent), adds the cleaning reagent to this row of biochips, and then the waste liquid extraction mechanism extracts the cleaning reagent waste liquid, then the pipette mechanism carries the pipette tip to the top of the reaction area, descends in the Z-axis height direction, releases the incubation reagent to the side of the biochip, waits for a predetermined time, such as 5 minutes, and sucks and pumps on the side multiple times, such as 5 times, and the pipette mechanism carries multiple pipette tips to the top of the
  • the waste liquid extraction component of the waste liquid extraction mechanism such as a steel needle, moves to the top of the biochip box, the steel needle descends, and the incubation reagent waste liquid is extracted from the same row of biochips at the same time.
  • the incubation step if it is determined that the biochips are arranged in three or more rows, the incubation step described above in which the multiple biochips are arranged in one row is sequentially performed on the three or more rows of biochips.
  • the cleaning step includes: determining whether the biochips are arranged in one row or two rows:
  • biochips are arranged in only one row, clean this row of biochips, take the pipette tip, add a certain amount of cleaning reagent with a lower concentration (for example, 0.1*SSC 400 ⁇ L), discard the pipette tip, and extract the waste liquid of the cleaning reagent.
  • a certain amount of cleaning reagent with a lower concentration for example, 0.1*SSC 400 ⁇ L
  • biochips are arranged in two rows, clean the biochips in the first row first, take the pipette tip, add a certain amount of cleaning reagent with a lower concentration (for example, 0.1*SSC 400 ⁇ L), discard the pipette tip, and extract the waste liquid of the cleaning reagent. Then clean the biochips in the second row, similar to the cleaning steps for the first row.
  • a certain amount of cleaning reagent with a lower concentration for example, 0.1*SSC 400 ⁇ L
  • the cleaning step if it is determined that the biochips are arranged in three or more rows, the cleaning step described above for multiple biochips arranged in one row is sequentially performed on the three or more rows of biochips.
  • the tissue permeabilization step includes: determining whether the biochips are arranged in one row or two rows in the biochip box:
  • Step S1 If the biochips are arranged in only one row. Add permeabilization reagent to the biochips in this row at the same time. After adding the permeabilization reagent, discard the pipette tip, cover the biochip box with a sealing cover and a temperature control cover, incubate at a certain temperature (e.g., 37°C), and open the sealing cover and the temperature control cover after the permeabilization time is over.
  • a certain temperature e.g. 37°C
  • the permeabilization time of the biochips in this row is the same, extract the waste liquid of the permeabilization reagent from the biochips in this row at the same time ⁇ add a certain amount of cleaning reagent with a lower concentration (e.g., 0.1*SSC) at the same time ⁇ extract the waste liquid of the cleaning reagent at the same time ⁇ add the reverse transcription reagent at the same time.
  • a certain amount of cleaning reagent with a lower concentration e.g., 0.1*SSC
  • Step S2 If the biochips are arranged in two rows, and the permeabilization time of the two rows of biochips is the same, add the permeabilization reagent (e.g., 400 ⁇ L) to the biochips in the first row for the first time. Add the permeabilization reagent (e.g., 400 ⁇ L) to the biochips in the second row for the second time. After adding the permeabilization reagent, discard the pipette tip, cover the biochip box with a sealing cover and a temperature control cover, incubate at a certain temperature (e.g., 37°C), and after the permeabilization time is over, open the sealing cover and the temperature control cover.
  • a certain temperature e.g., 37°C
  • a certain amount of cleaning reagent with a lower concentration e.g., 0.1*SSC
  • the tissue permeabilization step if it is determined that the biochips are arranged in three or more rows and the permeabilization time of all the biochips is the same, the three or more rows of biochips are divided into multiple groups of two adjacent rows of biochips, or into at least one group of two adjacent rows of biochips and one row of biochips.
  • Step S1 is performed on one row of biochips
  • step S2 is performed on each group of multiple groups of two adjacent rows of biochips or at least one group of two adjacent rows of biochips in sequence, that is, for each group of two adjacent rows of biochips, a permeabilization reagent is first added to the previous row of biochips, and then a permeabilization reagent is added to the next row of biochips, when the permeabilization time of the previous row of biochips ends, the permeabilization waste liquid of the previous row of biochips is extracted and a washing reagent is added at the same time, when the permeabilization time of the next row of biochips ends, the permeabilization waste liquid of the next row of biochips is extracted and a washing reagent is added at the same time, then the washing waste liquid of the previous row of biochips is extracted and a reverse transcription reagent is added at the same time, and then the washing waste liquid of the next row of biochips is extracted and a reverse transcription reagent is added at
  • the tissue permeabilization step includes: determining whether the biochips are arranged in one row or two rows:
  • Step S3 If the biochips are arranged in only one row in the biochip box. Add permeabilization reagent to the biochips in this row at the same time. After adding the permeabilization reagent, discard the pipette tip, cover the biochip box with a sealing cover and a temperature control cover, incubate at a certain temperature (for example, 37°C), and open the sealing cover and the temperature control cover after the permeabilization time is over. If the permeabilization time of the biochips in this row is not the same, and the difference in permeabilization time between each adjacent biochip with different permeabilization time is not less than twice the time of the entire single pipetting mechanism to draw liquid.
  • a certain temperature for example, 37°C
  • Step S4 When the biochips are arranged in two rows in the biochip box, the permeabilization time of the two rows of biochips is not the same, and the permeabilization time difference between each adjacent biochip with different permeabilization time is not less than twice the time of the single pipetting mechanism for the entire process of liquid extraction, and the permeabilization time of the first row of biochips is not the same as the permeabilization time of the second row of biochips.
  • the time difference between adding the permeabilization reagent to the first row of biochips and the second row of biochips is the time of the entire process of single liquid extraction.
  • the permeabilization reagent waste liquid is extracted from each biochip in the first row of biochips in turn ⁇ a certain amount of cleaning reagent with a lower concentration (for example, 0.1*SSC) is added ⁇ the cleaning reagent waste liquid is extracted ⁇ the reverse transcription reagent is added, and then the permeabilization reagent waste liquid is extracted from each biochip in the second row of biochips in turn ⁇ a certain amount of cleaning reagent with a lower concentration (for example, 0.1*SSC) is added ⁇ the cleaning reagent waste liquid is extracted ⁇ the reverse transcription reagent is added.
  • a certain amount of cleaning reagent with a lower concentration for example, 0.1*SSC
  • a plurality of biochips are arranged in two rows, the permeabilization time of the biochips in the two rows is not the same, and the permeabilization time difference between each adjacent biochip with different permeabilization time is not less than twice the time of the whole process of liquid extraction by a single pipetting mechanism, and the whole process of liquid extraction by a single pipetting mechanism is 1.5 minutes.
  • the first row includes the first to fourth biochips, and the second row includes the fifth to eighth biochips.
  • Step S5 first add permeabilization reagent to the biochip in the first row, and then add permeabilization reagent to the biochip in the second row. After adding the permeabilization reagent, discard the pipette tip, cover the biochip box with a sealing cover and a temperature control cover, incubate at a certain temperature (e.g., 37° C.), and open the sealing cover and the temperature control cover after the permeabilization time is over. Except for the fourth and fifth biochips, in the order of the end of the permeabilization time, the permeabilization reagent waste liquid is extracted, the cleaning reagent with a lower concentration (e.g., 0.1*SSC) is added, the cleaning agent waste liquid is extracted, and the reverse transcription reagent is added.
  • a lower concentration e.g., 0.1*SSC
  • the permeabilization time of the first and second biochips is 3 minutes, the above steps are performed synchronously on the first and second biochips. Similarly, if the permeabilization time of some or all biochips in a row is the same, some or all biochips are operated synchronously.
  • the permeabilization reagent waste liquid is extracted and the cleaning reagent with a lower concentration (e.g., 0.1*SSC) is added to the fourth biochip in the first row
  • the permeabilization reagent waste liquid is extracted and the cleaning reagent with a lower concentration (e.g., 0.1*SSC) is added to the fifth biochip in the second row
  • the cleaning agent waste liquid is extracted and the reverse transcription reagent is added to the fourth biochip in the first row
  • the cleaning agent waste liquid is extracted and the reverse transcription reagent is added to the fifth biochip in the second row.
  • the biochips are arranged in three or more rows, the permeabilization time of each biochip is not the same, and the difference in permeabilization time between two adjacent biochips with different permeabilization times is not less than twice the time of the entire process of liquid extraction by a single pipetting mechanism.
  • the three or more rows of biochips are divided into multiple groups of two adjacent rows of biochips, or into at least one group of two adjacent rows of biochips and one row of biochips.
  • Step S3 is performed on one row of biochips
  • step S4 or S5 is performed on each group of multiple groups of two adjacent rows of biochips or at least one group of two adjacent rows of biochips in sequence.
  • the reverse transcription step includes: determining whether the biochip is arranged in one row or two rows:
  • the biochip is arranged in only one row, add the reverse transcription mixed reagent to this row of biochips. After adding the reagent, discard the pipette tip, cover with a sealing cover and a temperature control cover, incubate at a certain temperature (e.g., 42°C), and after the time is up, open the sealing cover and the temperature control cover to extract the waste liquid of the reverse transcription mixed reagent.
  • a certain temperature e.g., 42°C
  • the biochips are arranged in two rows, add reagents to the biochips twice, first add reverse transcription mixed reagents to the biochips in the first row, and then add reverse transcription mixed reagents to the biochips in the second row. After adding the reagents, discard the pipette tip, cover with a sealing cover and a temperature control cover, and incubate at a certain temperature (e.g., 42°C). After the incubation time is up, open the sealing cover and the temperature control cover, and extract the waste liquid of the reverse transcription mixed reagent.
  • a certain temperature e.g. 42°C
  • reverse transcription mixed reagents are added to the three or more rows of biochips in sequence.
  • the pipette tip is discarded, and the sealing cover and the temperature control cover are added, and incubated at a certain temperature (e.g., 42° C.). After the time is up, the sealing cover and the temperature control cover are opened to extract the waste liquid of the reverse transcription mixed reagent.
  • the tissue removal step includes: determining whether the plurality of biochips are arranged in one row or two rows:
  • tissue removal reagent to this row of biochips. After adding the tissue removal reagent, discard the pipette tip, cover the biochip box with a sealing cover and a temperature control cover, and incubate at a certain temperature (for example, 55°C). After the incubation time is over, open the sealing cover and the temperature control cover, and draw out the tissue removal waste liquid.
  • a certain temperature for example, 55°C
  • the biochip is arranged in two rows, add the tissue removal reagent to the first row first, and then add the tissue removal reagent to the second row. After adding the tissue removal reagent, discard the pipette tip, cover the biochip box with a sealing cover and a temperature control cover, and incubate at a certain temperature (e.g., 55°C). After the incubation time is over, open the sealing cover and the temperature control cover to extract the waste liquid of the tissue removal reagent.
  • a certain temperature e.g. 55°C
  • the above-mentioned tissue removal step of only one row of biochips is sequentially performed on only three or more rows of biochips.
  • the nucleic acid release step includes: determining whether the biochips are arranged in one row or two rows:
  • the biochips are arranged in a row, add the nucleic acid release mixed reagent to the biochips in this row. After adding the nucleic acid release mixed reagent, discard the pipette tip, cover the biochip box with a sealing cover and a temperature control cover, and incubate at a certain temperature (e.g., 55° C.). After the incubation time is over, open the sealing cover and the temperature control cover to extract the waste liquid of the nucleic acid release mixed reagent.
  • a certain temperature e.g. 55° C.
  • the nucleic acid release mixed reagent is added to the first row first, and then to the second row.
  • the pipette tip is discarded, and the biochip box is covered with a sealing cover and a temperature control cover, and incubated at a certain temperature (e.g., 55° C.). After the incubation time is over, the sealing cover and the temperature control cover are opened to extract the waste liquid of the nucleic acid release mixed reagent.
  • the above-mentioned nucleic acid releasing step for only one row of biochips is sequentially performed on only three or more rows of biochips.
  • the product collection step includes: wherein the system controls the gripping mechanism to move to above the reaction area, opens the gripper, transfers the biochip box to the tilted area, and determines whether the biochips in the biochip box are in one row or two rows:
  • biochip If the biochip is arranged in only one row, all biochip products are collected at once.
  • the collection process is as follows: first take the pipette tip, the pipette mechanism carries the pipette tip and moves it to the top of the biochip on the inclined area.
  • the biochip box is inclined. First, the product at the lower position in the biochip box is extracted, and then the product at the higher position in the biochip box is extracted. Then the pipette mechanism reaches the product collection area, releases the collection reagent to the corresponding hole position, and throws the empty pipette tip into the trash can.
  • the above-mentioned product collecting step for the case where the biochips are arranged in only one row is performed sequentially for the biochips in the first row and the biochips in the second row.
  • the step of extracting waste liquid includes: determining whether the plurality of biochips are arranged in one row or two rows:
  • the waste liquid pumping mechanism is moved to the top of the biochip box, and the pump of the waste liquid pumping mechanism is started to first pump waste liquid from a higher position in the biochip box for a period of time (for example, 5 seconds), and then pump waste liquid from a lower position in the biochip box for a period of time (for example, 5 seconds), through the waste liquid pumping component, the waste liquid pumping component is raised along the Z axis and the pump is turned off, the waste liquid pumping mechanism is moved to the cleaning area, and the three-way valve is connected to clean the waste liquid pumping component for a period of time (for example, 3 seconds).
  • the waste liquid of the first row of biochip reactions is first sucked out, and then the waste liquid of the second row of biochip reactions is sucked out.
  • the suction step is the same as the above suction step when the multiple biochips are arranged in one row.
  • the above-mentioned product collecting step for the biochips arranged in only one row is sequentially performed for the three or more rows of biochips.
  • the waste liquid extraction mechanism may include a pump and a steel needle, or may include a pump and a disposable pipette tip.
  • the suction component of the waste liquid extraction mechanism may be a steel needle or a disposable pipette tip, and the pump may be an air pump, a rotary piston pump, a diaphragm pump or a plunger pump.
  • the execution phase includes testing operations, detecting the automated control method of the biochip and the reaction conditions of the automated control system that implements the automated control method, so as to better perform spatial omics capture and product collection.
  • the test operation uses steps similar to those of spatial omics, with the main differences being that a low-cost test biochip (such as a fluorescent biochip) is used, product collection is not required, and the tissue can be removed and cleaned before direct observation.
  • a low-cost test biochip such as a fluorescent biochip
  • the test operation uses steps similar to those of spatial omics, with the main differences being that a low-cost test biochip (such as a fluorescent biochip) is used, product collection is not required, and the material can be directly discharged for observation after tissue removal and cleaning.
  • a low-cost test biochip such as a fluorescent biochip
  • the permeabilization conditions of the test biochips in the same biochip box are set to be inconsistent. Specifically, the permeabilization times of the upper and lower rows of test biochips are different and some of the upper and lower rows of test biochips have the same permeabilization time, as shown in FIG. 7 .
  • whether the automated control method of the biochip and the reaction conditions of the automated control system implementing the automated control method need to be adjusted is determined based on the permeabilization effect observed after the material is loaded.
  • the size of the biochip carrying the target tissue can be customized, thereby improving the adaptability of the biochip.
  • the automated control method and automated control system disclosed herein are applied to multi-omics labeling, multi-omics capture and/or pathological labeling.
  • the following steps can be taken on the spatiotemporal biochip by an automated control system to achieve transcriptome capture and product collection.
  • the information of a single biochip is associated with the information of the biochip box by scanning the code, for example, manually inputting the permeabilization time of each biochip, and arranging multiple biochips in the biochip box in order from small to large permeabilization time, for example, arranging multiple biochips in at least one row.
  • manually placing execution consumables dispenser pipette tips, reagents, etc.
  • the execution starts with scanning the QR code on the outside of the biochip box and the biochip QR code in the biochip box to determine whether the biochip location, biochip quantity, and biochip QR code match the information in the system (manually entered/obtained from the docking system). If they do not match, a pop-up window will be displayed to remind the user: If the biochip information does not match the information in the system, the process will terminate; if they match, the next process will be entered;
  • the system When the code scanning is completed and the biochip information matches the system, the system will enter the state of the biochip box. If the existing biochip boxes in the system are not in the reverse transcription or nucleic acid release stage, the user will be reminded that it takes X time to transfer the biochip box to a reaction area. If all the biochip boxes in the system are in the reverse transcription or nucleic acid release step, the next process will be entered.
  • the biochip box Determine whether there are vacancies in the reaction area. If all reaction areas are occupied, the biochip box enters the waiting area and reminds the user: it takes a waiting time Y to transfer the biochip box to a reaction area. If there are vacancies in the reaction area, enter the next process.
  • the biochip box is clamped from the loading area by a grasping mechanism such as a clamp, and the biochip box is transferred to the reaction area, and then the following steps are performed in sequence: incubation step ⁇ cleaning step ⁇ tissue permeabilization step ⁇ reverse transcription step ⁇ cleaning step ⁇ tissue removal step ⁇ cleaning step ⁇ cDNA release step ⁇ cDNA collection step.
  • the present disclosure provides an automated control method for a biochip, wherein the automated control system of the biochip is used for labeling and capturing a proteome and collecting a product, and the labeling and capturing of a proteome and collecting a product include the following steps:
  • the information of a single biochip is associated with the information of the biochip box by scanning the code, for example, manually inputting the permeabilization time of each biochip, and arranging multiple biochips in the biochip box in order from small to large permeabilization time, for example, arranging multiple biochips in at least one row.
  • manually placing execution consumables dispenser pipette tips, reagents, etc.
  • the execution starts with scanning the QR code on the outside of the biochip box and the biochip QR code in the biochip box to determine whether the biochip location, biochip quantity, and biochip QR code match the information in the system (manually entered/obtained from the docking system). If they do not match, a pop-up window will be displayed to remind the user: If the biochip information does not match the information in the system, the process will terminate; if they match, the next process will be entered.
  • the system When the code scanning is completed and the biochip information matches the system, the system will enter the state of the biochip box. If the existing biochip boxes in the system are not in the reverse transcription or nucleic acid release stage, the user will be reminded that it takes X time to transfer the biochip box to a reaction area. If all the biochip boxes in the system are in the reverse transcription or nucleic acid release step, the next process will be entered.
  • the biochip box Determine whether there are vacancies in the reaction area. If all reaction areas are occupied, the biochip box enters the waiting area and reminds the user: it takes a waiting time Y to transfer the biochip box to a reaction area. If there are vacancies in the reaction area, enter the next process.
  • a biochip box is clamped from the loading area by a gripping mechanism such as a clamping claw, and the biochip box is transferred to the reaction area, and then the following steps are performed in sequence:
  • Cleaning Take a pipette tip, add a certain amount of cleaning reagent such as PBST and WB, clean the biochip, discard the pipette tip, and then extract the waste liquid of the cleaning reagent.
  • cleaning reagent such as PBST and WB
  • Blocking The blocking liquid is sucked by the pipetting mechanism, and then the pipetting mechanism carries the pipette tip that has sucked the blocking liquid to add the blocking liquid to the biochip. After the blocking is completed, the waste liquid of the blocking liquid is sucked away by the waste liquid extraction mechanism.
  • Antibody incubation The antibody reagent is sucked up by the pipetting mechanism, and then the pipetting mechanism carries the pipette tip that has sucked up the antibody reagent to add the antibody reagent to the biochip. After the incubation is completed, the waste liquid of the antibody reagent is sucked away by the waste liquid extraction mechanism.
  • Cleaning Take a pipette tip, add a certain amount of cleaning reagent such as PBST and WB, clean the biochip, discard the pipette tip, and then extract the waste liquid of the cleaning reagent.
  • cleaning reagent such as PBST and WB
  • Drying Drying the biochip in the biochip reaction area after the waste liquid is removed;
  • Proteome labeling and capture and product collection also include steps such as tissue permeabilization, reverse transcription, tissue removal, nucleic acid release, product collection, etc., which are the same as the corresponding steps of transcriptome capture and product collection.
  • the automated control method and automated control system of a biochip can bring at least one of the following technical advantages:
  • the entire execution process does not require human intervention.
  • the software interface can display the current execution steps and status in real time, and can also display the estimated remaining execution time.
  • the executors can predict and arrange the execution in advance according to the situation, saving manpower.
  • the present disclosure also provides an automated control system for automatically executing the steps in the above embodiments.

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Abstract

一种生物芯片的自动化控制方法及其自动化控制系统，自动化控制方法包括通过移液机构携带抽吸部件吸取试剂、通过移液机构携带已吸取试剂的抽吸部件向生物芯片加入试剂的操作，还提供了采用生物芯片的自动化控制方法的生物芯片自动化控制系统，生物芯片的自动化控制方法和系统可以对多张生物芯片自动调度与控制，从而完成对组织内生物信息的标记和捕获，并能对捕获产物执行整体收集，满足对生物芯片的高利用率高自动程度的处理要求。

Description

生物芯片的自动化控制方法及其自动化控制系统 技术领域
本公开涉及生物芯片的自动操作领域，特别涉及一种生物芯片的自动化控制方法及其自动化控制系统。
背景技术
空间转录组(Spatial Transcriptomics)是测量完整组织切片的总mRNA，将总mRNA的空间信息与形态学内容相结合，并绘制所有基因表达发生的位置，获得生物过程复杂而完整的基因表达图谱。可以结合显微成像和测序技术在获得基因表达数据的同时最大程度的保留样本的空间位置信息，为细胞功能、表型和组织微环境中位置的关系提供了重要信息。
空间转录组技术获取了空间位置和对应的基因表达信息，通过对空间转录组学数据执行分析，可以知道某些细胞是由哪些信号传导通路激活的。科学家们可通过空间转录组学技术产生的数据选择感兴趣的基因，并在原始的组织切片上显示其空间可辨的表达。由于捕获了所有mRNA，因此不再受限于只查看单个基因，而能够以任意组合的形式选择任意数量的基因来一起查看和分析。这些研究过程都需要通过生化反应将生物组织内的信息转换成可以探测的形式，其中涉及多种试剂以及通常需要繁琐的人工操作来重复多轮或者多步不同原理的生化反应。
发明人发现空间研究技术主要基于微流控芯片、激光显微切割、靶向多重染色、带寡聚核苷酸链修饰的捕获生物芯片等。其中10X基因组学主要提供基于手动操作的方案，提供配套仪器解决方案的有：Nano String公司的基于Leica的BondRX免疫组化染色仪和GeoMx平台执行多重染色和成像的激光捕获显微切割(Laser capture dissection)；Akoya公司基于PhenoCycler-Fusion的多重染色和原位成像。以上这些或者基于半自动的生化操作+显微成像+测序，或者基于全自动的显微镜和手动的生化操作，或者基于单张生物芯片的全自动多重染色+显微成像，均难以在生化反应自动程度高、空间分辨率高、样品处理通量大、捕获范围广等方面兼顾。
此外，发明人发现空间组学配套仪器主要集中于多重染色，或染色后的成像，或两者结合，这些仪器只能处理25mm*75mm长宽的标准生物芯片，不能兼容其它尺寸生物芯片的应用。针对其他平面生物芯片也不能适配。同时只能挑选感兴趣的目标区域收集目标物，难以实现产物的大规模全部收集。例如，不能针对厘米级别长宽的平板生物芯片 执行整体产物收集的功能。
另外，发明人发现包含收集样品执行混合测序的方案需要人工耗时挑选感兴趣的目标区域(ROI)，只能以较低通量执行，不能执行生物芯片、高通量的处理。发明人发现的仪器普遍需要目标生物芯片或生物芯片需要有额外用于密封和/或夹持的区域，不能同时兼顾生物芯片的高利用率和试剂耗量最小化。需要人员中途干预。
发明内容
为实现上述目的，本公开提供一种生物芯片的自动化控制方法，包括通过移液机构携带抽吸部件吸取试剂、通过移液机构携带已吸取试剂的抽吸部件向生物芯片加入试剂的操作。
在一些实施例中，操作包括以下步骤中的至少一个：
孵育：通过移液机构携带抽吸部件吸取孵育试剂，通过移液机构携带已吸取孵育试剂的抽吸部件向生物芯片加入孵育试剂；
组织透化：通过移液机构携带抽吸部件吸取透化试剂，通过移液机构携带已吸取透化试剂的抽吸部件向生物芯片加入透化试剂；
反转录：通过移液机构携带抽吸部件吸取反转录试剂，通过移液机构携带已吸取反转录试剂的抽吸部件向生物芯片加入反转录试剂；
组织去除：通过移液机构携带抽吸部件吸取组织去除试剂，通过移液机构携带已吸取组织去除试剂的抽吸部件向生物芯片加入组织去除试剂。
在一些实施例中，在操作的各步骤中，通过抽废液机构将废液抽走。
在一些实施例中，在组织去除步骤之后，还包括以下步骤中的至少一个：
核酸释放：通过移液机构携带抽吸部件吸取核酸释放试剂，通过移液机构携带已吸取核酸释放试剂的抽吸部件向生物芯片加入核酸释放试剂，完成核酸释放；
产物收集：通过移液机构携带抽吸部件来吸取释放的核酸，并保存释放的核酸。
在一些实施例中，在孵育步骤和组织透化步骤之间执行清洗步骤和/或在组织透化步骤和反转录步骤之间执行清洗步骤和/或在反转录步骤和组织去除步骤之间执行清洗步骤和/或在组织去除步骤和核酸释放步骤之间执行清洗步骤。
在一些实施例中，清洗步骤为：通过移液机构携带抽吸部件吸取清洗试剂，通过移液机构携带已吸取清洗试剂的抽吸部件向生物芯片加入清洗试剂，通过抽废液机构将清洗废液抽走。
在一些实施例中，操作的各步骤自动地执行。
在一些实施例中，在操作的各步骤中，实时向用户显示当前执行步骤和/或当前状态。
在一些实施例中，在操作之前，将多个生物芯片布置在生物芯片盒中，通过扫描将多个生物芯片的信息与生物芯片盒的信息关联在一起，并将多个生物芯片的透化时间输入系统。
在一些实施例中，提供载有目标组织的生物芯片，其尺寸是定制化的。
在一些实施例中，在生物芯片盒中将多个生物芯片依次排列成至少一行。
在一些实施例中，如果多个生物芯片的透化时间均相同：
在多个生物芯片被排列为一行时，则对一行生物芯片同时抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂；
在多个生物芯片被排列成二行时，则先给第一行生物芯片抽透化试剂废液，加清洗试剂，再给第二行生物芯片抽透化试剂废液，加清洗试剂，然后给第一行生物芯片抽清洗试剂废液，加反转录试剂，再然后给第二行生物芯片抽清洗试剂废液，加反转录试剂。
在一些实施例中，如果多个生物芯片的透化时间不均相同，并且多个生物芯片按照透化时间从小到大的顺序依次排列,
在多个生物芯片被排列成一行时，按照透化时间完成的先后顺序，依次对多个生物芯片抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂；
在多个生物芯片被排列成二行时，并且两行生物芯片之间没有相同的透化时间，按照透化时间完成的先后顺序，先依次对第一行生物芯片的各生物芯片抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂，再依次对第二行生物芯片的各生物芯片抽透化试剂废液→加清洗试剂→抽清洗试剂废液→加反转录试剂；以及
在多个生物芯片被排列成二行时，如果两行生物芯片之间存在相同的透化时间，按照透化时间结束的先后顺序，先依次对第一行生物芯片的除了具有该相同时间的生物芯片之外的其他生物芯片抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂，在第一行生物芯片的具有该相同时间的生物芯片抽透化试剂废液并加清洗试剂之后，对第二行生物芯片的具有该相同时间的生物芯片抽透化试剂废液并加清洗试剂，然后对第一行生物芯片的具有该相同时间的生物芯片抽清洗试剂废液并加反转录试剂，接着对第二行生物芯片的具有该相同时间的生物芯片抽清洗试剂废液并加反转录试剂，然后依次对第二行生物芯片的其他生物芯片抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂。
在一些实施例中，自动化控制方法包括抽废液步骤，抽废液步骤包括以下步骤：
在多个生物芯片布置为一行时，抽废液机构移动到生物芯片盒上方，启动所述抽废 液机构的泵通过废液抽吸部件先抽取生物芯片盒中位于较高位置的废液、再抽取生物芯片盒中位于较低位置的废液，沿着Z轴升高废液抽吸部件并关闭泵，抽废液机构移动到清洗区进行清洗；以及
在所述多个生物芯片被排列成二行时，先抽吸第一行生物芯片反应的废液，再抽吸第二行生物芯片反应的废液，抽吸步骤与所述多个生物芯片布置为一行的抽吸步骤相同。
在一些实施例中，在输入各生物芯片的透化时间之后，通过系统扫描生物芯片盒的信息和生物芯片盒中的生物芯片的信息，判断生物芯片在生物芯片盒中的位置、生物芯片的数量、生物芯片的信息与系统里的信息是否匹配：
如果不匹配，则提醒用户；
如果匹配，则判断系统中已存在的生物芯片盒的状态：
如果已存在的生物芯片盒有不处于反转录步骤或核酸释放步骤，则提醒用户还需要等待时长X才能将生物芯片盒转移到反应区以执行操作；以及
如果已存在的生物芯片盒均处于反转录步骤或核酸释放步骤，则判断反应区是否有空位，如果没有空位，则提醒用户还需等待时长Y才能将生物芯片盒转移到反应区，如果有空位，则将生物芯片盒转移到反应区以执行操作。
在一些实施例中，在将生物芯片置于生物芯片盒的步骤和操作之间，通过抓取机构将生物芯片盒转移到反应区以执行操作，抓取机构与移液机构是彼此独立工作的。
在一些实施例中，在操作的步骤中，抓取机构和/或移液机构均不触碰生物芯片。
在一些实施例中，通过抓取机构对生物芯片盒执行加盖密封盖和/或控温盖以及打开密封盖和/或控温盖。
在一些实施例中，在产物收集步骤中，通过抓取机构先将生物芯片盒转移到倾斜区，并在倾斜区完成产物收集。
在一些实施例中，在产物收集步骤中，由移液机构携带抽吸部件吸取试剂，由移液机构携带已吸取试剂的抽吸部件对生物芯片盒中的生物芯片执行冲洗，用移液机构携带抽吸部件执行产物收集。
在一些实施例中，在操作之前包括通过移液机构取抽吸部件的步骤，该步骤包括：判断抽吸部件容器中的当前列是否符合取抽吸部件条件，当数量和位置都满足条件时，移液机构移动到该列抽吸部件上方，高度下降，同时在该列取抽吸部件，如果当前列不满足取抽吸部件条件时，轮询下一列，直到该抽吸部件容器轮询完，开始轮询下一个抽吸部件容器，直到轮询完所有抽吸部件容器，如果还没有满足取抽吸部件条件的列，则提醒用户补充抽吸部件。
在一些实施例中，移液机构吸取试剂的步骤包括：移液机构携带抽吸部件转移到试剂区上方，高度下降，探测试剂液面，如果未探测到试剂，则提醒用户补充试剂，如果探测到试剂，则判断富余量是否满足所携带的抽吸部件要吸取的量，如果否，则提醒用户补充试剂，如果是，进一步判断要添加的试剂是否为清洗试剂，如果是清洗试剂，则所有抽吸部件的高度下降，一起吸取清洗试剂，如果不是清洗试剂，则各抽吸部件的高度分别下降，吸取反应试剂。
在一些实施例中，所述自动化控制方法被应用于转录组学、蛋白组学、代谢组学和/或脂质组学的标记或捕获以及病理标记。
本公开还提供一种生物芯片的自动化控制系统，所述自动化控制系统包括生物芯片盒、反应区、移液机构和抓取机构，并且被构造成生物芯片的上述自动化控制方法。
本公开所提供的自动化控制方法和自动化控制系统可以实现对多个生物芯片可扩展的自动调度与控制。实现对组织内生物信息的标记和捕获、以及转录组、蛋白组、免疫组等多组学的分析，并实现完成捕获产物的收集功能。
本公开所提供的方法和系统满足多个生物芯片、高利用率、高通量、高自动程度的处理要求。
附图说明
图1为本公开的生物芯片的自动化控制方法的整体流程图；
图2为本公开的生物芯片的自动化控制方法的取移液枪头的流程图；
图3为本公开的生物芯片的自动化控制方法的加试剂的流程图；
图4为本公开的生物芯片的自动化控制方法的孵育步骤的流程图；
图5为本公开的生物芯片的自动化控制方法的清洗步骤的流程图；
图6为本公开的生物芯片的自动化控制方法的组织透化步骤的流程图；
图7为本公开的具有不同透化时间的两行生物芯片的示意图；
图8为本公开的生物芯片的自动化控制方法的反转录步骤的流程图；
图9为本公开的生物芯片的自动化控制方法的组织去除步骤的流程图；
图10为本公开的生物芯片的自动化控制方法的核酸释放步骤的流程图；
图11为本公开的生物芯片的自动化控制方法的收集步骤的流程图；以及
图12为本公开的生物芯片的自动化控制方法的抽废液步骤的流程图。
具体实施方式
下面结合具体实施方式对本公开执行进一步的详细描述，给出的实施例仅为了阐明本公开，而不是为了限制本公开的范围。
本公开提供一种用于生物芯片的自动化控制方法以及实施该自动化控制方法的自动化控制系统。
如图1所示，本公开的自动化控制方法包括执行开始前阶段和执行阶段。
在执行开始前阶段，可以操作以下步骤：
在系统之外，通过扫码将单个生物芯片与生物芯片盒绑定起来，并输入每张生物芯片的透化时间，例如通过手动输入。
放置执行耗材(抽吸部件例如移液枪头、试剂等)，例如通过人工放置。
选择软件界面的执行流程，并点击开始按钮，进入自动运行的流程：
从扫码开始，扫描生物芯片盒外侧的二维码，并扫描生物芯片盒里的生物芯片二维码，判断生物芯片位置/生物芯片数/生物芯片二维码与系统里的(手动录入/从对接系统获取)信息是否匹配。如果不匹配，则软件弹窗提醒用户：生物芯片信息与系统里的信息不匹配，流程终止；如果匹配，则进入下一流程。
扫码完成并且生物芯片信息与系统匹配时，程序进入判断系统里生物芯片盒状态的流程。如果系统里的生物芯片盒有不处在反转录或核酸释放阶段，则弹窗提醒用户：还需等待时间X才可以转移生物芯片盒到反应区。如果系统里的生物芯片盒均处于反转录或核酸释放阶段，则进入下一判断流程。
判断反应区是否有空位，如果各反应区均被占用，则该生物芯片盒进入等待区，并弹窗提醒用户：还需等待时间Y才可以转移生物芯片盒到反应区。如果反应区有空位，则程序进入下一流程。
控制抓取机构从上料区夹取生物芯片盒，并转移到反应区，抓取机构与移液机构是独立工作的。可选地，抓取机构包括夹爪，移液机构包括多通道移液机械臂。
系统根据所选执行流程，执行生化反应。
本公开的自动化控制方法只在执行开始前阶段，需要人为输入执行信息，放置执行耗材和试剂。整个执行阶段无需人为干预，软件界面可实时显示当前的执行步骤和状态，并可显示预估剩余执行时间，操作人员可根据情况提前预知并安排操作，节省人力。
在执行阶段实现空间组学(例如转录组学、蛋白组学、代谢组学和脂质组学)的捕获和产物收集，执行阶段包括以下步骤：
在一些实施例中，如图2所示，取移液枪头步骤包括：判断当前列是否符合取移液枪头条件，当且仅当数量和位置都符合时，满足条件，移液机构移动到该列移液枪头上方， Z轴(高度方向)下降，同时在该列取移液枪头。如果当前列不满足取移液枪头条件时，轮询下一列，直到该移液枪头盒轮询完，开始轮询下一个移液枪头盒，直到轮询完所有移液枪头盒，如果还没有满足取移液枪头条件的列，则弹窗提醒用户：请补充移液枪头。
在一些实施例中，如图3所示，添加试剂步骤包括：判断生物芯片盒中的生物芯片是一行或两行：
如果生物芯片排列为仅一行，则对一行生物芯片一次加液即可。
如果生物芯片排列为两行，先给第一行生物芯片加液：给第一行生物芯片取移液枪头，移液机构携带移液枪头转移到试剂区上方，Z轴(高度方向)下降，探测试剂液面，如果未探测到试剂，则弹窗提醒用户：请补充试剂。如果探测到，则判断富余量是否够所携带移液枪头要吸取的量，如果不够，则弹窗提醒用户：请补充试剂。如果富余量足够，判断要添加的试剂是否为清洗试剂，如果是清洗试剂，则所有Z轴(高度方向)下降，一起吸取清洗试剂，清洗试剂装载大容量的容器中，如果不是清洗试剂，则Z轴(高度方向)分别下降，吸取反应试剂，反应试剂位于小容量的孔板中。吸取反应试剂后，移液机构携带移液枪头转移到在反应区的生物芯片盒上方，Z轴(高度方向)下降，加反应试剂到生物芯片侧边。接着给第二行生物芯片加液，与第一行生物芯片的加液步骤相同。
在一些实施例中，在添加试剂步骤中，如果生物芯片被排列为三行或更多行，依次对三行或更多行生物芯片执行对生物芯片被排列为仅一行的上述添加试剂步骤。
在一些实施例中，如图4所示，孵育步骤包括：判断多个生物芯片在生物芯片盒中被排列成一行还是二行：
如果生物芯片被排列为仅一行，则给这行生物芯片取移液枪头(取移液枪头流程如图3所示)，移液机构例如多通道移液机械臂携带移液枪头去试剂区，抽取一定量浓度较高(例如5*SSC 400μL)的清洗试剂(抽取试剂流程见图3)，将清洗试剂加这行生物芯片，然后抽废液机构将清洗试剂废液抽出，接着移液机构携带多个移液枪头去反应区上方，在Z轴(高度方向)方向上下降，吐孵育试剂到生物芯片侧方，等待预定时间例如5分钟后，在侧边吸打多次例如5次，移液机构携带多个移液枪头去垃圾桶上方，丢弃移液枪头。加完孵育试剂后，抽废液机构的抽吸部件例如钢针走到生物芯片盒上方，钢针下降，给这行生物芯片抽孵育试剂废液。
如果多个生物芯片被排列成二行，则先给第一行生物芯片取移液枪头(取移液枪头流程见图3)，移液机构例如多通道移液机械臂携带移液枪头去试剂区，抽取一定量浓度较高(例如5*SSC 400μL)的清洗试剂(抽取试剂流程见图3)，将清洗试剂加这行生物芯片，然后抽废液机构将清洗试剂废液抽出，接着移液机构携带移液枪头去反应区上方， 在Z轴高度方向上下降，释放孵育试剂到生物芯片侧方，等预定时间例如5分钟后，在侧边吸打多次例如5次，移液机构携带多个移液枪头去垃圾桶上方，丢弃移液枪头。接着给第二行生物芯片取移液枪头，重复上述步骤。加完孵育试剂后，抽废液机构的抽废液部件例如钢针走到生物芯片盒上方，钢针下降，同一行生物芯片同时抽孵育试剂废液。
在一些实施例中，在孵育步骤中，如果判断生物芯片被排列为三行或更多行，依次对三排或更多排生物芯片执行上述多个生物芯片被排列成一行的孵育步骤。
在一些实施例中，如图5所示，清洗步骤包括：判断生物芯片被排列为一行还是二行：
如果生物芯片被排列为仅一行，则给这行生物芯片清洗，取移液枪头，加一定量浓度较低(例如0.1*SSC 400μL)的清洗试剂，丢弃移液枪头，抽清洗试剂废液。
如果生物芯片被排列为两行，先给第一行生物芯片清洗，取移液枪头，加一定量浓度较低(例如0.1*SSC 400μL)的清洗试剂，丢弃移液枪头，抽清洗试剂废液。再给第二行生物芯片清洗，与第一行的清洗步骤类似。
在一些实施例中，在清洗步骤中，如果判断生物芯片被排列为三行或更多行，依次对三排或更多排生物芯片执行上述多个生物芯片被排列成一行的清洗步骤。
在一些实施例中，如图6所示，组织透化步骤包括：判断生物芯片在生物芯片盒中被排列为一行还是两行：
步骤S1：如果生物芯片被排列为仅一行。给这行生物芯片同时加透化试剂，加完透化试剂后，丢弃移液枪头，给生物芯片盒加盖密封盖、温控盖，在一定温度(例如37℃)下温育，透化时间结束后，打开密封盖、温控盖。如果这行生物芯片的透化时间都相同，给这行生物芯片同时抽透化试剂废液→同时加一定量浓度较低(例如0.1*SSC)清洗试剂→同时抽清洗试剂废液→同时加反转录试剂。
步骤S2：如果生物芯片被排列为两行，且两行生物芯片的透化时间均相同，第一次先给第一行生物芯片加透化试剂(例如400μL)。第二次给第二行生物芯片加透化试剂(例如400μL)。加完透化试剂后，丢弃移液枪头，给生物芯片盒加盖密封盖、温控盖，在一定温度(例如37℃)下温育，透化时间结束后，打开密封盖、温控盖。先给第一行抽透化试剂废液，加一定量浓度较低(例如0.1*SSC)的清洗试剂，再给第二行生物芯片抽清洗试剂废液，加一定量浓度较低(例如0.1*SSC)的清洗试剂，然后从第一行抽清洗试剂废液，加反转录试剂，接着从第二行生物芯片抽清洗试剂废液，加反转录试剂。
在一些实施例中，在组织透化步骤中，如果判断生物芯片被排列成三行或更多行，且所有生物芯片的透化时间均相同，将三行或更多行生物芯片分成多组相邻两行生物芯片，或者分成至少一组相邻两行生物芯片和一行生物芯片。对一行生物芯片执行步骤S1， 依次对多组相邻两行生物芯片或至少一组相邻两行生物芯片中的每组执行步骤S2，即对于每组相邻两行生物芯片，先对前一行生物芯片加透化试剂，再对后一行生物芯片加透化试剂，当前一行生物芯片的透化时间结束时，抽取前一行生物芯片反应的透化废液并同时加清洗试剂，当后一行生物芯片的透化时间结束时，抽取后一行生物芯片反应的透化废液并同时加清洗试剂，接着对前一行生物芯片抽清洗废液并同时加反转录试剂，然后对后一行生物芯片抽清洗废液并同时加反转录试剂。
在一些实施例中，如图6所示，组织透化步骤包括：判断生物芯片被排列为一行还是两行：
步骤S3：如果生物芯片在生物芯片盒中被排列为仅一行。给这行生物芯片同时加透化试剂加完透化试剂后，丢弃移液枪头，给生物芯片盒加盖密封盖、温控盖，在一定温度(例如37℃)下温育，透化时间结束后，打开密封盖、温控盖。如果这行生物芯片的透化时间不都相同，并且每相邻且透化时间不同的两个生物芯片之间的透化时间差均不小于两倍的单次移液机构抽注液全程的时间。按照透化时间结束的先后顺序，依次对这行生物芯片的各生物芯片抽透化试剂废液→加浓度较低(例如0.1*SSC)的清洗试剂→抽清洗试剂废液→加反转录试剂。
步骤S4：当生物芯片在生物芯片盒中被排列为二行时，两行生物芯片的透化时间不都相同，并且每相邻且透化时间不同的两个生物芯片之间的透化时间差均不小于两倍的单次移液机构抽注液全程的时间，第一行生物芯片的各透化时间与第二行生物芯片的各透化时间没有相同的。先给第一行生物芯片加透化试剂，再给第二次给第二行生物芯片加透化试剂，给第一行生物芯片与第二行生物芯片加透化试剂的时间差为单次抽注液全程的时间。加完透化试剂后，丢弃移液枪头，给生物芯片盒加盖密封盖、温控盖，在一定温度(例如37℃)下温育，经过透化时间之后，打开密封盖、温控盖。按照透化时间结束的先后顺序，依次对第一行生物芯片的各生物芯片抽透化试剂废液→加一定量浓度较低(例如0.1*SSC)的清洗试剂→抽清洗剂废液→加反转录试剂，再给依次对第二行生物芯片的各生物芯片抽透化试剂废液→加一定量浓度较低(例如0.1*SSC)的清洗试剂→抽清洗试剂废液→反转录试剂。
在一些实施例中，如图7所示，多个生物芯片被排列为两行，两行生物芯片的透化时间不都相同，并且每相邻且透化时间不同的两个生物芯片之间的透化时间差均不小于两倍的单次移液机构抽注液全程的时间，单次移液机构抽注液全程的时间为1.5分钟。第一行包括第一到第四生物芯片，第二行包括第五到第八生物芯片。第一行和第二行生物芯片中有相同透化时间的生物芯片，第一行的第四个生物和第二行的第五个生物芯片的透 化时间均为9min。步骤S5：先给第一行生物芯片加透化试剂，再给第二行生物芯片加透化试剂。加完透化试剂后，丢弃移液枪头，给生物芯片盒加盖密封盖、温控盖，在一定温度(例如37℃)下温育，透化时间结束后，打开密封盖、温控盖。除了第四个和第五个生物芯片之外，按照透化时间结束的前后顺序，依次对第一行生物芯片的各生物芯片抽透化试剂废液、加浓度较低(例如0.1*SSC)的清洗试剂、抽清洗剂废液和加反转录试剂。由于第一个和第二个生物芯片的透化时间都为3min，对第一个和第二个生物芯片同步执行上述步骤。类似地，如果一排中有一些或全部生物芯片的透化时间相同，则对一些或全部生物芯片同步操作。在对第一行的第四个生物芯片执行抽透化试剂废液并加浓度较低(例如0.1*SSC)的清洗试剂之后，再对第二行的第五个生物芯片抽透化试剂废液并加浓度较低(例如0.1*SSC)的清洗试剂，然后再对第一行的第四个生物芯片抽清洗剂废液并加反转录试剂，接着对第二行的第五个生物芯片执行抽清洗剂废液并加反转录试剂。
在一些实施例中，生物芯片被排列为三行或更多行，各生物芯片的透化时间不都相同，并且每相邻且透化时间不同的两个生物芯片之间的透化时间差均不小于两倍单次移液机构抽注液全程的时间。将三行或更多行生物芯片分成多组相邻两行生物芯片，或者分成至少一组相邻两行生物芯片和一行生物芯片。对一行生物芯片执行步骤S3，依次对多组相邻两行生物芯片或者至少一组相邻两行生物芯片中的每组执行步骤S4或S5。
在一些实施例中，如图8所示，反转录步骤包括：判断生物芯片被排列为一行或两行:
如果生物芯片被排列为仅一行，则给这行生物芯片加反转录混合试剂。加完试剂后，丢弃移液枪头，加盖密封盖、温控盖，在一定温度下(例如42℃)温育，时间到后，打开密封盖、温控盖，抽反转录混合试剂废液。
如果生物芯片被排列为两行，则分两次给生物芯片加试剂，先给第一行生物芯片加反转录混合试剂，给第二行生物芯片加反转录混合试剂。加完试剂后，丢弃移液枪头，加盖密封盖、温控盖，在一定温度(例如42℃)下温育，温育时间到后，打开密封盖、温控盖，抽反转录混合试剂废液。
在一些实施例中，在反转录步骤中，如果生物芯片被排列为三行或更多行，依次对三行或更多行生物芯片加反转录混合试剂。加完反转录混合试剂后，丢弃移液枪头，加盖密封盖、温控盖，在一定温度下(例如42℃)温育，时间到后，打开密封盖、温控盖，抽反转录混合试剂废液。
在一些实施例中，如图9所示，组织去除步骤包括：判断多个生物芯片被排列为一行或两行:
如果生物芯片被排列为仅一行，给这行生物芯片加组织去除试剂，加完组织去除试剂后，丢弃移液枪头，对生物芯片盒加盖密封盖、温控盖，在一定温度(例如55℃)下温育，温育时间结束后，打开密封盖、温控盖，抽组织去除废液。
如果生物芯片被排列为两行，先给第一行加组织去除试剂，再给第二行加组织去除试剂。加完组织去除试剂后，丢弃移液枪头，给生物芯片盒加盖密封盖、温控盖，在一定温度(例如55℃)下温育，温育时间结束后，打开密封盖、温控盖，抽组织去除试剂废液。
在一些实施例中，在组织去除步骤中，如果生物芯片被排列为仅三行或更多行，依次对仅三行或更多行的生物芯片执行仅一行生物芯片的上述组织去除步骤。
在一些实施例中，如图10所示，核酸释放步骤包括：判断生物芯片被排列为一行或两行:
如果生物芯片被排列为一行，则给这行生物芯片加核酸释放混合试剂。加完核酸释放混合试剂后，丢弃移液枪头，对生物芯片盒加盖密封盖、温控盖，在一定温度(例如55℃)下温育，温育时间结束后，打开密封盖、温控盖，抽核酸释放混合试剂废液。
如果将生物芯片排列为两行，先给第一行加核酸释放混合试剂，再给第二行加核酸释放混合试剂。加完核酸释放混合试剂后，丢弃移液枪头，对生物芯片盒加盖密封盖、温控盖，在一定温度(例如55℃)下例如温育，温育时间结束后，打开密封盖、温控盖，抽核酸释放混合试剂废液。
在一些实施例中，在核酸释放步骤中，如果生物芯片被排列为仅三行或更多行，依次对仅三行或更多行的生物芯片执行仅一行生物芯片的上述核酸释放步骤。
在一些实施例中，如图11所示，产物收集步骤包括：其中系统控制抓取机构移动到反应区上方，张开夹爪，转移生物芯片盒到倾斜区，判断生物芯片盒中的生物芯片为一行还是两行：
如果生物芯片被排列为仅一行，则一次收集所有的生物芯片产物。收集流程如下：先取移液枪头，移液机构携带移液枪头移动到在倾斜区上的生物芯片上方，生物芯片盒是倾斜的，先抽位于生物芯片盒中较低位置的产物，再抽位于生物芯片盒中较高位置的产物，然后移液机构到达产物收集区，将收集试剂释放到对应孔位，将空移液枪头丢到垃圾桶，再取移液枪头，加一定量(例如400μL)无核酸酶水清洗，收集清洗产物，先抽位于生物芯片盒中较低位置的产物，再抽位于生物芯片盒中较高位置的产物，然后移液机构到达产物收集区，将收集试剂释放到对应孔位，再将空移液枪头丢到垃圾桶。
如果生物芯片被排列为两行，依次对第一行生物芯片和第二行生物芯片执行对生物 芯片被排列为仅一行的上述产物收集步骤。
在一些实施例中，如图12所示，抽废液步骤包括：判断多个生物芯片布置为一行或两行：
如果多个生物芯片布置为一行，抽废液机构移动到生物芯片盒上方，启动抽废液机构的泵通过废液抽吸部件先在生物芯片盒中从位于较高位置抽吸一段时间(例如5秒)废液、再在生物芯片盒中从位于较低位置抽吸一段时间(例如5秒)的废液，沿着Z轴升高废液抽吸部件并关闭泵，抽废液机构移动到清洗区，接通三通阀清洗抽废液部件一段时间(例如3秒)。
如果多个生物芯片被排列成二行，先抽吸第一行生物芯片反应的废液，再抽吸第二行生物芯片反应的废液，抽吸步骤与所述多个生物芯片布置为一行的上述抽吸步骤相同。
在一些实施例中，在产物收集步骤中，如果生物芯片被排列为三行或更多行，依次对三行或更多行生物芯片执行对生物芯片被排列为仅一行的上述产物收集步骤。
在一些实施例中，抽废液机构可以包括泵和钢针，也可以包括泵和一次性移液枪头，抽废液机构的抽吸部件可以为钢针或一次性移液枪头，泵可以是空气泵、旋转柱塞泵、隔膜泵或柱塞泵。
在一些实施例中，在执行阶段包括测试操作，检测生物芯片的自动化控制方法以及实施该自动化控制方法的自动化控制系统的反应条件，以更好地进行空间组学的捕获和产物收集。
在一些实施例中，测试操作采用与空间组学类似的步骤，主要区别在于：使用成本低廉的测试生物芯片(例如荧光生物芯片)，不需要进行产物收集，组织去除清洗后直接下料观察。
在一些实施例中，测试操作采用与空间组学类似的步骤，主要区别在于：使用成本低廉的测试生物芯片(例如荧光生物芯片)，不需要进行产物收集，组织去除清洗后直接下料观测。
在一些实施例中，在测试操作中，设定同一生物芯片盒中测试生物芯片的透化条件不一致，具体地，上下两行测试生物芯片透化时间有不同且存在上下两行部分测试生物芯片透化时间相同，例如图7所示。
在一些实施例中，根据下料观测到的透化效果判断是否需要调整生物芯片的自动化控制方法以及实施该自动化控制方法的自动化控制系统的反应条件。
在一些实施例中，载有目标组织的生物芯片的尺寸可以定制。由此，从而提高了生物芯片的适配灵活性。
在一些实施例中，据本公开的自动化控制方法和自动化控制系统应用于多组学标记、多组学捕获和/或病理标记。在一些实施例中，如图1所示，可以通过自动化控制系统对时空生物芯片采取以下步骤实现转录组捕获和产物收集。
在系统之外，通过扫码单个生物芯片的信息与生物芯片盒的信息关联起来，例如手动输入各生物芯片的透化时间，并且按照透化时间从小到大的顺序，依次将多个生物芯片排列在生物芯片盒中，例如将多个生物芯片至少排列为至少一行。通过例如手动放置执行耗材(一次性移液枪头、试剂等)。选择软件界面的执行流程，并点击开始按钮，系统进入自动运行的流程。
执行从扫码开始，扫描生物芯片盒外侧的二维码，并扫描生物芯片盒里的生物芯片二维码，判断生物芯片位置、生物芯片数量、生物芯片二维码与系统里的(手动录入/从对接系统获取)信息是否匹配。如果不匹配，则弹窗提醒用户：如果生物芯片信息与系统里的信息不匹配，则流程终止；如果匹配，则进入下一流程；
扫码完成并且生物芯片信息与系统匹配时，进入判断系统里生物芯片盒状态。如果系统里的已经存在的生物芯片盒有不处在反转录或核酸释放阶段，则提醒用户：还需等待时长X可以转移生物芯片盒到某个反应区。如果系统里的生物芯片盒均处于反转录或核酸释放步骤，则进入下一流程。
判断反应区是否有空位，如果各反应区均被占用，则生物芯片盒进入等待区，并提醒用户：还需等待时长Y可以转移该生物芯片盒到某个反应区。如果反应区有空位，则进入下一流程。
如图1所示，通过抓取机构例如夹爪从上料区夹取生物芯片盒，并将生物芯片盒转移到反应区，然后依次执行以下上述步骤：孵育步骤→清洗步骤→组织透化步骤→反转录步骤→清洗步骤→组织去除步骤→清洗步骤→cDNA释放步骤→cDNA收集步骤。
在一些实施例中，本公开提供一种生物芯片的自动化控制方法，将生物芯片的自动化控制系统用于蛋白质组的标记和捕获以及产物收集，实现蛋白质组的标记和捕获以及产物收集包括以下操作步骤：
在系统之外，通过扫码单个生物芯片的信息与生物芯片盒的信息关联起来，例如手动输入各生物芯片的透化时间，并且按照透化时间从小到大的顺序，依次将多个生物芯片排列在生物芯片盒中，例如将多个生物芯片至少排列为至少一行。通过例如手动放置执行耗材(一次性移液枪头、试剂等)。选择软件界面的执行流程，并点击开始按钮，系统进入自动运行的流程。
执行从扫码开始，扫描生物芯片盒外侧的二维码，并扫描生物芯片盒里的生物芯片 二维码，判断生物芯片位置、生物芯片数量、生物芯片二维码与系统里的(手动录入/从对接系统获取)信息是否匹配。如果不匹配，则弹窗提醒用户：如果生物芯片信息与系统里的信息不匹配，则流程终止；如果匹配，则进入下一流程
扫码完成并且生物芯片信息与系统匹配时，进入判断系统里生物芯片盒状态。如果系统里的已经存在的生物芯片盒有不处在反转录或核酸释放阶段，则提醒用户：还需等待时长X可以转移生物芯片盒到某个反应区。如果系统里的生物芯片盒均处于反转录或核酸释放步骤，则进入下一流程。
判断反应区是否有空位，如果各反应区均被占用，则生物芯片盒进入等待区，并提醒用户：还需等待时长Y可以转移该生物芯片盒到某个反应区。如果反应区有空位，则进入下一流程。
如图1所示，通过抓取机构例如夹爪从上料区夹取生物芯片盒，并将生物芯片盒转移到反应区，然后依次执行以下步骤：
清洗：取移液枪头，加一定量清洗试剂例如PBST和WB，给生物芯片清洗，丢移液枪头，然后抽清洗试剂废液。
封闭：通过移液机构吸取封闭液，再由移液机构携带已吸取封闭液的移液枪头向生物芯片加入封闭液，封闭完成后再由抽废液机构将封闭液废液抽走。
抗体孵育：通过移液机构吸取抗体试剂，再由移液机构携带已吸取抗体试剂的移液枪头向生物芯片加入抗体试剂，孵育完成后再由抽废液机构将抗体试剂废液抽走。
清洗：取移液枪头，加一定量清洗试剂例如PBST和WB，给生物芯片清洗，丢移液枪头，然后抽清洗试剂废液。
干燥：将抽走废液后的生物芯片在生物芯片反应区中执行干燥；
蛋白质组的标记和捕获以及产物收集还包括组织透化、反转录、组织去除、核酸释放、产物收集等步骤，这些步骤与转录组捕获和产物收集的相应步骤相同。
根据本公开的各个实施例，生物芯片的自动化控制方法和自动化控制系统能够带来以下技术优点中的至少一个：
整个执行过程无须人为干预。
软件界面可实时显示当前执行步骤和状态，并可显示预估剩余执行时间，执行人员可根据情况提前预知并安排执行，节省人力。
可以同时处理多个全生物芯片样本，单日通量高。
能够兼顾生物芯片的高利用率和试剂量消耗的最小化。
实现百分百的生物芯片表面利用率。以及
支持定制化尺寸的生物芯片，完成平面生物芯片的生化反应和表面产物的分离和收集。
本公开还提供用于自动执行以上实施例中的步骤的自动化控制系统。
最后应说明的是：以上实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述实施例对本公开执行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案执行修改，或者对其中部分技术特征执行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的精神和范围。

Claims (24)

  1. 一种生物芯片的自动化控制方法，包括通过移液机构携带抽吸部件吸取试剂、通过所述移液机构携带已吸取所述试剂的抽吸部件向生物芯片加入试剂的操作。
  2. 根据权利要求1所述的生物芯片的自动化控制方法，其中所述操作包括以下步骤中的至少一个：
    孵育：通过移液机构携带抽吸部件吸取孵育试剂，通过所述移液机构携带已吸取孵育试剂的抽吸部件向所述生物芯片加入孵育试剂；
    组织透化：通过所述移液机构携带抽吸部件吸取透化试剂，通过所述移液机构携带已吸取透化试剂的抽吸部件向所述生物芯片加入透化试剂；
    反转录：通过所述移液机构携带抽吸部件吸取反转录试剂，通过所述移液机构携带已吸取反转录试剂的抽吸部件向所述生物芯片加入反转录试剂；
    组织去除：通过所述移液机构携带抽吸部件吸取组织去除试剂，通过所述移液机构携带已吸取组织去除试剂的抽吸部件向所述生物芯片加入组织去除试剂。
  3. 根据权利要求2所述的生物芯片的自动化控制方法，其中在所述操作的各步骤中，通过抽废液机构将废液抽走。
  4. 根据权利要求2所述的生物芯片的自动化控制方法，其中在组织去除步骤之后，还包括以下步骤中的至少一个：
    核酸释放：通过所述移液机构携带抽吸部件吸取核酸释放试剂，通过所述移液机构携带已吸取核酸释放试剂的抽吸部件向所述生物芯片加入核酸释放试剂，完成核酸释放；
    产物收集：通过所述移液机构携带抽吸部件来吸取释放的核酸，并保存释放的核酸。
  5. 根据权利要求4所述的生物芯片的自动化控制方法，其中在所述孵育步骤和所述组织透化步骤之间执行清洗步骤和/或在所述组织透化步骤和所述反转录步骤之间执行清洗步骤和/或在所述反转录步骤和所述组织去除步骤之间执行清洗步骤和/或在所述组织去除步骤和所述核酸释放步骤之间执行清洗步骤。
  6. 根据权利要求5所述的生物芯片的自动化控制方法，其中所述清洗步骤为：通过所述移液机构携带抽吸部件吸取清洗试剂，通过所述移液机构携带已吸取清洗试剂的抽吸部件向所述生物芯片加入清洗试剂，通过所述抽废液机构将清洗废液抽走。
  7. 根据权利要求2-6中任一项所述的生物芯片的自动化控制方法，其中所述操作的各步骤自动地执行。
  8. 根据权利要求2-7中任一项所述的生物芯片的自动化控制方法，其中在所述操作的 各步骤中，实时向用户显示当前执行步骤和/或当前状态。
  9. 根据权利要求1-8中任一项所述的生物芯片的自动化控制方法，其中在所述操作之前，将多个生物芯片布置在生物芯片盒中，通过扫描将所述多个生物芯片的信息与所述生物芯片盒的信息关联在一起，并将所述多个生物芯片的透化时间输入系统。
  10. 根据权利要求1-9中任一项所述的生物芯片的自动化控制方法，其中提供载有目标组织的生物芯片，其尺寸是定制化的。
  11. 根据权利要求2-10中任一项所述的生物芯片的自动化控制方法，其中在所述生物芯片盒中将所述多个生物芯片依次排列成至少一行。
  12. 根据权利要求11所述的生物芯片的自动化控制方法，其特征在于，如果所述多个生物芯片的透化时间均相同：
    在所述多个生物芯片被排列为一行时，则对一行生物芯片同时抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂；
    在所述多个生物芯片被排列成二行时，则先给第一行生物芯片抽透化试剂废液，加清洗试剂，再给第二行生物芯片抽透化试剂废液，加清洗试剂，然后给第一行生物芯片抽清洗试剂废液，加反转录试剂，再然后给第二行生物芯片抽清洗试剂废液，加反转录试剂。
  13. 根据权利要求11所述的生物芯片的自动化控制方法，其中如果所述多个生物芯片的透化时间不均相同，并且所述多个生物芯片按照透化时间从小到大的顺序依次排列,
    在所述多个生物芯片被排列成一行时，按照透化时间完成的先后顺序，依次对所述多个生物芯片抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂；
    在所述多个生物芯片被排列成二行时，并且两行生物芯片之间没有相同的透化时间，按照透化时间完成的先后顺序，先依次对第一行生物芯片的各生物芯片抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂，再依次对第二行生物芯片的各生物芯片抽透化试剂废液→加清洗试剂→抽清洗试剂废液→加反转录试剂；以及
    在所述多个生物芯片被排列成二行时，如果两行生物芯片之间存在相同的透化时间，按照透化时间结束的先后顺序，先依次对第一行生物芯片的除了具有该相同时间的生物芯片之外的其他生物芯片抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂，在第一行生物芯片的具有该相同时间的生物芯片抽透化试剂废液并加清洗试剂之后，对第二行生物芯片的具有该相同时间的生物芯片抽透化试剂废液并加清洗试剂，然后对第一行生物芯片的具有该相同时间的生物芯片抽清洗试剂废液并加反转录试剂，接着对第二行生物芯片的具有该相同时间的生物芯片抽清洗试剂废液并加反转录试剂，然 后依次对第二行生物芯片的其他生物芯片抽透化试剂废液→加清洗试剂→抽清洗剂废液→加反转录试剂。
  14. 根据权利要求12或13所述的生物芯片的自动化控制方法，包括抽废液步骤，所述抽废液步骤包括：
    在所述多个生物芯片布置为一行时，抽废液机构移动到生物芯片盒上方，启动所述抽废液机构的泵通过废液抽吸部件先抽取生物芯片盒中位于较高位置的废液、再抽取生物芯片盒中位于较低位置的废液，沿着Z轴升高废液抽吸部件并关闭泵，抽废液机构移动到清洗区进行清洗；以及
    在所述多个生物芯片被排列成二行时，先抽吸第一行生物芯片反应的废液，再抽吸第二行生物芯片反应的废液，抽吸步骤与所述多个生物芯片布置为一行的抽吸步骤相同。
  15. 根据权利要求9-14中任一项所述的生物芯片的自动化控制方法，其中在输入各生物芯片的透化时间之后，通过系统扫描所述生物芯片盒的信息和所述生物芯片盒中的所述生物芯片的信息，判断所述生物芯片在所述生物芯片盒中的位置、所述生物芯片的数量、所述生物芯片的信息与系统里的信息是否匹配：
    如果不匹配，则提醒用户；
    如果匹配，则判断系统中已存在的生物芯片盒的状态：
    如果所述已存在的生物芯片盒有不处于反转录步骤或核酸释放步骤，则提醒用户还需要等待时长X才能将所述生物芯片盒转移到所述反应区以执行所述操作；以及
    如果所述已存在的生物芯片盒均处于反转录步骤或核酸释放步骤，则判断所述反应区是否有空位，如果没有空位，则提醒用户还需等待时长Y才能将所述生物芯片盒转移到所述反应区，如果有空位，则将所述生物芯片盒转移到所述反应区以执行所述操作。
  16. 根据权利要求10-15中任一所述的生物芯片的自动化控制方法，其中在将所述生物芯片置于所述生物芯片盒的步骤和所述操作之间，通过抓取机构将所述生物芯片盒转移到反应区以执行所述操作，所述抓取机构与所述移液机构是彼此独立工作的。
  17. 根据权利要求16所述的生物芯片的自动化控制方法，其中在所述操作的步骤中，所述抓取机构和/或所述移液机构均不触碰所述生物芯片。
  18. 根据权利要求16或17所述的生物芯片的自动化控制方法，其中通过所述抓取机构对所述生物芯片盒执行加盖密封盖和/或控温盖以及打开密封盖和/或控温盖。
  19. 根据权利要求4所述的生物芯片的自动化控制方法，其中在所述产物收集步骤中， 通过抓取机构先将所述生物芯片盒转移到倾斜区，并在所述倾斜区完成产物收集。
  20. 根据权利要求4或19所述的生物芯片的自动化控制方法，其中在所述产物收集步骤中，由所述移液机构携带抽吸部件吸取试剂，由所述移液机构携带已吸取试剂的抽吸部件对所述生物芯片盒中的所述生物芯片执行冲洗，用所述移液机构携带抽吸部件执行产物收集。
  21. 根据权利要求1-20中任一所述的生物芯片的自动化控制方法，其中在所述操作之前包括通过所述移液机构取抽吸部件的步骤，该步骤包括：判断抽吸部件容器中的当前列是否符合取抽吸部件条件，当数量和位置都满足条件时，所述移液机构移动到该列抽吸部件上方，高度下降，同时在该列取抽吸部件，如果当前列不满足取抽吸部件条件时，轮询下一列，直到该抽吸部件容器轮询完，开始轮询下一个抽吸部件容器，直到轮询完所有抽吸部件容器，如果还没有满足取抽吸部件条件的列，则提醒用户补充抽吸部件。
  22. 根据权利要求1-21中任一所述的生物芯片的自动化控制方法，其中所述移液机构吸取试剂的步骤包括：所述移液机构携带抽吸部件转移到试剂区上方，高度下降，探测试剂液面，如果未探测到试剂，则提醒用户补充试剂，如果探测到试剂，则判断富余量是否满足所携带的抽吸部件要吸取的量，如果否，则提醒用户补充试剂，如果是，进一步判断要添加的试剂是否为清洗试剂，如果是清洗试剂，则所有所述抽吸部件的高度下降，一起吸取清洗试剂，如果不是清洗试剂，则各所述抽吸部件的高度分别下降，吸取反应试剂。
  23. 根据权利要求1-22中任一项所述的生物芯片的自动化控制方法，其中所述自动化控制方法被应用于转录组学、蛋白组学、代谢组学和/或脂质组学的标记或捕获以及病理标记。
  24. 一种生物芯片的自动化控制系统，其中所述自动化控制系统包括生物芯片盒、反应区、移液机构和抓取机构，并且被构造成用于执行权利要求1-23中任一项所述的生物芯片的自动化控制方法。
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