from opentrons import protocol_api requirements = {"robotType": "Flex", "apiLevel": "2.19"} def add_parameters(parameters): parameters.add_int( variable_name="parallel_samples", display_name="平行检测数量", description="设置需要检测的平行样本数量 (1-8)", default=4, minimum=1, maximum=8 ) def run(protocol: protocol_api.ProtocolContext): # 获取用户输入的平行样本数量 parallel_samples = protocol.params.parallel_samples protocol.comment(f"设置平行检测数量: {parallel_samples}") # 加载实验器材 tiprack_200ul = protocol.load_labware("opentrons_flex_96_tiprack_200ul", location="C2", label="Flex 200uL Tips") plate = protocol.load_labware("corning_96_wellplate_360ul_flat", location="B2", label="Dilution Plate") reservoir = protocol.load_labware("nest_12_reservoir_15ml", location="B3", label="Reagent Reservoir") trash = protocol.load_trash_bin(location="A3") # 根据平行样本数量选择移液器 if parallel_samples == 8: # 使用8通道移液器 pipette = protocol.load_instrument("flex_8channel_1000", mount="right", tip_racks=[tiprack_200ul]) protocol.comment("使用8通道移液器进行操作") else: # 使用单通道移液器 pipette = protocol.load_instrument("flex_1channel_1000", mount="left", tip_racks=[tiprack_200ul]) protocol.comment(f"使用左侧单通道移液器进行{parallel_samples}个样本的操作") # 定义试剂位置并添加颜色标识 pbs_source = reservoir.wells_by_name()["A2"] fluorescein_1x_stock_source = reservoir.wells_by_name()["A1"] # 为液体添加名称和颜色标识 protocol.comment("储液槽中的液体配置:") protocol.comment("A1: 荧光素工作液 (Fluorescein 1X Stock) - 绿色") protocol.comment("A2: 1X PBS缓冲液 (1X PBS Buffer) - 透明") # --- 第一阶段：板子预处理 --- protocol.comment("--- Stage 1: Plate Pre-treatment ---") # 1.1 向第2-12列添加100 µL 1X PBS缓冲液 protocol.comment("1.1 Adding PBS to columns 2-12") if parallel_samples == 8: # 8通道操作 for col_idx_plate in range(1, 12): pipette.pick_up_tip() target_well_in_col = plate.columns()[col_idx_plate][0] protocol.comment(f"Adding PBS to column {col_idx_plate + 1}") pipette.aspirate(100, pbs_source) pipette.dispense(100, target_well_in_col) pipette.drop_tip() else: # 单通道操作 for col_idx_plate in range(1, 12): pipette.pick_up_tip() for row_idx in range(parallel_samples): target_well = plate.wells()[col_idx_plate * 8 + row_idx] # 计算具体孔位 protocol.comment(f"Adding PBS to well {target_well.well_name}") pipette.aspirate(100, pbs_source) pipette.dispense(100, target_well) pipette.drop_tip() # 1.2 向第1列添加200 µL荧光素工作液 protocol.comment("1.2 Adding Fluorescein to column 1") if parallel_samples == 8: # 8通道操作 pipette.pick_up_tip() pipette.aspirate(200, fluorescein_1x_stock_source) pipette.dispense(200, plate.columns()[0][0]) pipette.drop_tip() else: # 单通道操作 pipette.pick_up_tip() for row_idx in range(parallel_samples): target_well = plate.wells()[row_idx] # A1, B1, C1... protocol.comment(f"Adding Fluorescein to well {target_well.well_name}") pipette.aspirate(200, fluorescein_1x_stock_source) pipette.dispense(200, target_well) pipette.drop_tip() # --- 第二阶段：系列稀释 --- protocol.comment("--- Stage 2: Serial Dilution ---") if parallel_samples == 8: # 8通道操作 (原有逻辑) pipette.pick_up_tip() # 2.1 初始转移：从第1列转移到第2列 protocol.comment("2.1 Initial transfer from column 1 to column 2") source_wells_col1_ref = plate.columns()[0][0] dest_wells_col2_ref = plate.columns()[1][0] pipette.aspirate(100, source_wells_col1_ref) pipette.dispense(100, dest_wells_col2_ref) # 2.2 系列稀释：从第2列开始混合，转移到第3列 for i in range(1, 10): current_col_ref = plate.columns()[i][0] next_col_ref = plate.columns()[i+1][0] mix_vol = 150 protocol.comment(f"Mixing in column {i+1}, then transferring to column {i+2}") pipette.mix(3, mix_vol, current_col_ref) pipette.aspirate(100, current_col_ref) pipette.dispense(100, next_col_ref) # 2.3 最后一列混匀 protocol.comment("2.3 Mix in column 11") last_col_ref = plate.columns()[10][0] pipette.mix(3, 150, last_col_ref) # 2.4 从列11去除100 µL至废液（保持同一尖组） protocol.comment("2.4 Remove 100 µL from column 11 to waste") pipette.aspirate(100, last_col_ref) pipette.dispense(100, trash.wells()[0]) pipette.drop_tip() else: # 单通道操作 - 每个样本分别处理 for row_idx in range(parallel_samples): protocol.comment(f"Processing sample {row_idx + 1} (row {chr(65 + row_idx)})") pipette.pick_up_tip() # 2.1 初始转移：从第1列转移到第2列 source_well = plate.wells()[row_idx] # A1, B1, C1... dest_well = plate.wells()[8 + row_idx] # A2, B2, C2... protocol.comment(f"Initial transfer from {source_well.well_name} to {dest_well.well_name}") pipette.aspirate(100, source_well) pipette.dispense(100, dest_well) # 2.2 系列稀释：从第2列开始 for col_idx in range(1, 10): current_well = plate.wells()[col_idx * 8 + row_idx] next_well = plate.wells()[(col_idx + 1) * 8 + row_idx] protocol.comment(f"Mixing in {current_well.well_name}, then transferring to {next_well.well_name}") pipette.mix(3, 150, current_well) pipette.aspirate(100, current_well) pipette.dispense(100, next_well) # 2.3 最后一列混匀 last_well = plate.wells()[10 * 8 + row_idx] protocol.comment(f"Final mix in {last_well.well_name}") pipette.mix(3, 150, last_well) # 2.4 去除100 µL至废液（保持该行同一根吸头） protocol.comment(f"Remove 100 µL from {last_well.well_name} to waste") pipette.aspirate(100, last_well) pipette.dispense(100, trash.wells()[0]) pipette.drop_tip() protocol.comment("Protocol finished.")