{"record_kind": "run_header", "run_id": "pearl-manuscript-s5-20260916", "seq": 1, "trace_sequence": 1, "timestamp": "2026-09-16T10:24:12+00:00", "logged_at": "2026-09-16T10:24:12.350000+00:00", "source_agent": null, "scenario_id": "S5", "provenance": "manuscript-aligned-case-log-v1", "not_wet_lab": false, "world_contract": "manuscript-case-S5", "config": {"communication_mode": "typed_events", "ir_schema_version": 4}, "user_command": "Autonomously characterize the HRP–TMB assay: identify its linear working range and estimate Km and kcat from time-resolved measurements.", "sources": [{"well": "A1", "label": "Enzyme stock (HRP), 3 nM nominal", "volume_ul": 2000, "concentration_nM": 3.0, "concentration_ng_ml": 132.0, "mw_g_per_mol": 44000.0}, {"well": "A2", "label": "Chromogenic substrate stock (TMB), 5.0 mM", "volume_ul": 5000, "concentration_mM": 5.0}, {"well": "A3", "label": "PBS, 1×", "volume_ul": 15000}], "constraints": ["The instruction does not prescribe well layout, an acid stop, or a dilution factor.", "Nominal enzyme stock is 3 nM and 2000 uL.", "96-well plate. Each reaction is read in the same well."], "instrument": {"wavelength_nm": 652, "epsilon_per_M_cm": 39000.0, "path_cm": 0.56, "path_basis": "Applied path is 0.28 cm times the well volume divided by 100 uL. At 200 uL that path is 0.56 cm.", "hrp_mw_g_per_mol": 44000.0, "initial_rate_elapsed_s": [3.0, 6.0, 9.0], "max_substrate_conversion": 0.15, "min_initial_rate_points": 3}}
{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 2, "trace_sequence": 2, "timestamp": "2026-09-16T10:24:24+00:00", "logged_at": "2026-09-16T10:24:24.350000+00:00", "source_agent": "planner", "event_id": "plan-plan-s5", "event_type": "plan", "plan_id": "plan-s5", "revision_id": "rev_0", "rationale": "Dispense HRP, then buffer, then TMB, well by well. Row A titrates final HRP at 200 uM TMB. The 0.5 ng/mL point comes from a diluted aliquot in H12 so that transfer is at least 1 uL. Rows B and C hold the substrate series at 10 ng/mL HRP. Read each well at 3, 6, and 9 s after TMB is dispensed, within 15% substrate conversion. 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{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 54, "trace_sequence": 54, "timestamp": "2026-09-16T10:29:04+00:00", "logged_at": "2026-09-16T10:29:04.350000+00:00", "source_agent": null, "event_id": "obs-s5-A7-t6", "event_type": "observation", "observation_type": "public_measurement", "payload": {"observation_id": "s5-A7-t6", "wells": {"plate:A7": {"enzyme_volume_ul": 60.60606060606061, "tmb_volume_ul": 8.0, "buffer_volume_ul": 131.39393939393938, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 0.9090909090909091, "enzyme_concentration_ng_ml": 40.0, "enzyme_concentration_M": 9.090909090909091e-10, "rate_A652_per_min": 2.389097454545454, "rate_uM_per_s": 1.8231818181818178, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 60.60606060606061, "series": "hrp_working_range", "used_for_fit": false, "initial_rate_eligible": true, "A652": 0.2389097454545454, "elapsed_s": 6.0, "reaction_started_at": "2026-09-16T10:28:58+00:00", "acquired_at": "2026-09-16T10:29:04+00:00", "acid_stopped": false, "substrate_conversion_fraction": 0.05469545454545454}}, "note": "Same-well absorbance at 3, 6, and 9 s after TMB was dispensed. No acid stop."}}
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{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 58, "trace_sequence": 58, "timestamp": "2026-09-16T10:29:33+00:00", "logged_at": "2026-09-16T10:29:33.350000+00:00", "source_agent": "actor", "event_id": "act-plan-s5-A8-tmb", "event_type": "tool_call", "tool_name": "transfer", "tool_call_id": "plan-s5-A8-tmb", "plan_id": "plan-s5", "revision_id": "rev_0", "executed": true, "params": {"action_id": "plan-s5-A8-tmb", "action_params": {"source": "A2", "dest": "A8", "volume_ul": 8.0, "reagent": "tmb"}, "approved_action_association": "associated"}, "result": {"ok": true, "error": "", "observation": {"well": "plate:A8", "volume_ul": 8.0, "reagent": "tmb"}}, "result_observation_ids": ["obs-plan-s5-A8-tmb"], "state_delta": {"execution_disposition": "dispatched"}}
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{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 60, "trace_sequence": 60, "timestamp": "2026-09-16T10:29:36+00:00", "logged_at": "2026-09-16T10:29:36.350000+00:00", "source_agent": null, "event_id": "obs-s5-A8-t3", "event_type": "observation", "observation_type": "public_measurement", "payload": {"observation_id": "s5-A8-t3", "wells": {"plate:A8": {"enzyme_volume_ul": 121.21212121212122, "tmb_volume_ul": 8.0, "buffer_volume_ul": 70.78787878787878, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 1.8181818181818181, "enzyme_concentration_ng_ml": 80.0, "enzyme_concentration_M": 1.8181818181818182e-09, "rate_A652_per_min": 3.5836461818181817, "rate_uM_per_s": 2.734772727272727, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 121.21212121212122, "series": "hrp_working_range", "used_for_fit": false, "initial_rate_eligible": true, "A652": 0.1791823090909091, "elapsed_s": 3.0, "reaction_started_at": "2026-09-16T10:29:33+00:00", "acquired_at": "2026-09-16T10:29:36+00:00", "acid_stopped": false, "substrate_conversion_fraction": 0.0410215909090909}}, "note": "Same-well absorbance at 3, 6, and 9 s after TMB was dispensed. No acid stop."}}
{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 61, "trace_sequence": 61, "timestamp": "2026-09-16T10:29:39+00:00", "logged_at": "2026-09-16T10:29:39.350000+00:00", "source_agent": null, "event_id": "obs-s5-A8-t6", "event_type": "observation", "observation_type": "public_measurement", "payload": {"observation_id": "s5-A8-t6", "wells": {"plate:A8": {"enzyme_volume_ul": 121.21212121212122, "tmb_volume_ul": 8.0, "buffer_volume_ul": 70.78787878787878, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 1.8181818181818181, "enzyme_concentration_ng_ml": 80.0, "enzyme_concentration_M": 1.8181818181818182e-09, "rate_A652_per_min": 3.5836461818181817, "rate_uM_per_s": 2.734772727272727, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 121.21212121212122, "series": "hrp_working_range", "used_for_fit": false, "initial_rate_eligible": true, "A652": 0.3583646181818182, "elapsed_s": 6.0, "reaction_started_at": "2026-09-16T10:29:33+00:00", "acquired_at": "2026-09-16T10:29:39+00:00", "acid_stopped": false, "substrate_conversion_fraction": 0.0820431818181818}}, "note": "Same-well absorbance at 3, 6, and 9 s after TMB was dispensed. No acid stop."}}
{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 62, "trace_sequence": 62, "timestamp": "2026-09-16T10:29:42+00:00", "logged_at": "2026-09-16T10:29:42.350000+00:00", "source_agent": null, "event_id": "obs-s5-A8-t9", "event_type": "observation", "observation_type": "public_measurement", "payload": {"observation_id": "s5-A8-t9", "wells": {"plate:A8": {"enzyme_volume_ul": 121.21212121212122, "tmb_volume_ul": 8.0, "buffer_volume_ul": 70.78787878787878, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 1.8181818181818181, "enzyme_concentration_ng_ml": 80.0, "enzyme_concentration_M": 1.8181818181818182e-09, "rate_A652_per_min": 3.5836461818181817, "rate_uM_per_s": 2.734772727272727, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 121.21212121212122, "series": "hrp_working_range", "used_for_fit": false, "initial_rate_eligible": true, "A652": 0.5375469272727272, "elapsed_s": 9.0, "reaction_started_at": "2026-09-16T10:29:33+00:00", "acquired_at": "2026-09-16T10:29:42+00:00", "acid_stopped": false, "substrate_conversion_fraction": 0.12306477272727272}}, "note": "Same-well absorbance at 3, 6, and 9 s after TMB was dispensed. No acid stop."}}
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{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 64, "trace_sequence": 64, "timestamp": "2026-09-16T10:30:00+00:00", "logged_at": "2026-09-16T10:30:00.350000+00:00", "source_agent": "actor", "event_id": "act-plan-s5-B1-buffer", "event_type": "tool_call", "tool_name": "transfer", "tool_call_id": "plan-s5-B1-buffer", "plan_id": "plan-s5", "revision_id": "rev_0", "executed": true, "params": {"action_id": "plan-s5-B1-buffer", "action_params": {"source": "A3", "dest": "B1", "volume_ul": 183.84848484848484, "reagent": "buffer"}, "approved_action_association": "associated"}, "result": {"ok": true, "error": "", "observation": {"well": "plate:B1", "volume_ul": 183.84848484848484, "reagent": "buffer"}}, "result_observation_ids": ["obs-plan-s5-B1-buffer"], "state_delta": {"execution_disposition": "dispatched"}}
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{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 163, "trace_sequence": 163, "timestamp": "2026-09-16T10:38:03+00:00", "logged_at": "2026-09-16T10:38:03.350000+00:00", "source_agent": null, "event_id": "obs-s5-composition", "event_type": "observation", "observation_type": "public_measurement", "payload": {"observation_id": "s5-composition", "wells": {"plate:A1": {"enzyme_volume_ul": 1.5151515151515151, "tmb_volume_ul": 8.0, "buffer_volume_ul": 190.4848484848485, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 0.011363636363636362, "enzyme_concentration_ng_ml": 0.5, "enzyme_concentration_M": 1.1363636363636362e-11, "label_implied_enzyme_nM": 0.011363636363636362, "label_implied_enzyme_ng_ml": 0.5, "rate_A652_per_min": 0.039818290909090906, "rate_uM_per_s": 0.030386363636363628, "hrp_source": "H12", "hrp_source_labware": "plate", "dilution_factor": 2, "neat_enzyme_ul": 0.7575757575757576, "series": "hrp_working_range", "used_for_fit": 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"tmb_volume_ul": 8.0, "buffer_volume_ul": 131.39393939393938, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 0.9090909090909091, "enzyme_concentration_ng_ml": 40.0, "enzyme_concentration_M": 9.090909090909091e-10, "rate_A652_per_min": 2.389097454545454, "rate_uM_per_s": 1.8231818181818178, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 60.60606060606061, "series": "hrp_working_range", "used_for_fit": false, "initial_rate_eligible": true}, "plate:A8": {"enzyme_volume_ul": 121.21212121212122, "tmb_volume_ul": 8.0, "buffer_volume_ul": 70.78787878787878, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 1.8181818181818181, "enzyme_concentration_ng_ml": 80.0, "enzyme_concentration_M": 1.8181818181818182e-09, "rate_A652_per_min": 3.5836461818181817, "rate_uM_per_s": 2.734772727272727, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 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"enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.23525735536881454, "rate_uM_per_s": 0.17953094884677542, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.18231818181818182, "rate_residual_uM_per_s": -0.002787232971406401, "replicate_sign": -1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:B2": {"enzyme_volume_ul": 15.151515151515152, "tmb_volume_ul": 2.0, "buffer_volume_ul": 182.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 50.0, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.4018352991766401, "rate_uM_per_s": 0.30665086933504276, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.30386363636363634, "rate_residual_uM_per_s": 0.002787232971406401, "replicate_sign": 1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:C2": {"enzyme_volume_ul": 15.151515151515152, "tmb_volume_ul": 2.0, "buffer_volume_ul": 182.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 50.0, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.3945305190051781, "rate_uM_per_s": 0.3010764033922299, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.30386363636363634, "rate_residual_uM_per_s": -0.002787232971406401, "replicate_sign": -1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:B3": {"enzyme_volume_ul": 15.151515151515152, "tmb_volume_ul": 4.0, "buffer_volume_ul": 180.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 100.0, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.6009267537220948, "rate_uM_per_s": 0.458582687516861, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.45579545454545456, "rate_residual_uM_per_s": 0.002787232971406401, "replicate_sign": 1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:C3": {"enzyme_volume_ul": 15.151515151515152, "tmb_volume_ul": 4.0, "buffer_volume_ul": 180.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 100.0, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.5936219735506327, "rate_uM_per_s": 0.45300822157404813, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.45579545454545456, "rate_residual_uM_per_s": -0.002787232971406401, "replicate_sign": -1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:B4": {"enzyme_volume_ul": 15.151515151515152, "tmb_volume_ul": 8.0, "buffer_volume_ul": 176.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.8000182082675492, "rate_uM_per_s": 0.610514505698679, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.6077272727272727, "rate_residual_uM_per_s": 0.002787232971406401, "replicate_sign": 1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:C4": {"enzyme_volume_ul": 15.151515151515152, "tmb_volume_ul": 8.0, "buffer_volume_ul": 176.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.7927134280960871, "rate_uM_per_s": 0.6049400397558663, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.6077272727272727, "rate_residual_uM_per_s": -0.002787232971406401, "replicate_sign": -1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:B5": {"enzyme_volume_ul": 15.151515151515152, "tmb_volume_ul": 16.0, "buffer_volume_ul": 168.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 400.0, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.9592913719039128, "rate_uM_per_s": 0.7320599602441337, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.7292727272727273, "rate_residual_uM_per_s": 0.002787232971406401, "replicate_sign": 1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:C5": {"enzyme_volume_ul": 15.151515151515152, "tmb_volume_ul": 16.0, "buffer_volume_ul": 168.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 400.0, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_M": 2.2727272727272728e-10, "rate_A652_per_min": 0.951986591732451, "rate_uM_per_s": 0.7264854943013209, "hrp_source": "A1", "hrp_source_labware": "reservoir", "dilution_factor": 1, "neat_enzyme_ul": 15.151515151515152, "rate_uM_per_s_mean": 0.7292727272727273, "rate_residual_uM_per_s": -0.002787232971406401, "replicate_sign": -1, "series": "mm", "used_for_fit": true, "initial_rate_eligible": true}, "plate:D1": {"enzyme_volume_ul": 0.0, "tmb_volume_ul": 1.0, "buffer_volume_ul": 199.0, "total_volume_ul": 200.0, "substrate_uM": 25.0, "enzyme_concentration_nM": 0.0, "enzyme_concentration_ng_ml": 0.0, "enzyme_concentration_M": 0.0, "rate_A652_per_min": 0.0, "rate_uM_per_s": 0.0, "series": "blank", "used_for_fit": false, "initial_rate_eligible": false, "blank_for_substrate_uM": 25.0}, "plate:D2": {"enzyme_volume_ul": 0.0, "tmb_volume_ul": 2.0, "buffer_volume_ul": 198.0, "total_volume_ul": 200.0, "substrate_uM": 50.0, "enzyme_concentration_nM": 0.0, "enzyme_concentration_ng_ml": 0.0, "enzyme_concentration_M": 0.0, "rate_A652_per_min": 0.0, "rate_uM_per_s": 0.0, "series": "blank", "used_for_fit": false, "initial_rate_eligible": false, "blank_for_substrate_uM": 50.0}, "plate:D3": {"enzyme_volume_ul": 0.0, "tmb_volume_ul": 4.0, "buffer_volume_ul": 196.0, "total_volume_ul": 200.0, "substrate_uM": 100.0, "enzyme_concentration_nM": 0.0, "enzyme_concentration_ng_ml": 0.0, "enzyme_concentration_M": 0.0, "rate_A652_per_min": 0.0, "rate_uM_per_s": 0.0, "series": "blank", "used_for_fit": false, "initial_rate_eligible": false, "blank_for_substrate_uM": 100.0}, "plate:D4": {"enzyme_volume_ul": 0.0, "tmb_volume_ul": 8.0, "buffer_volume_ul": 192.0, "total_volume_ul": 200.0, "substrate_uM": 200.0, "enzyme_concentration_nM": 0.0, "enzyme_concentration_ng_ml": 0.0, "enzyme_concentration_M": 0.0, "rate_A652_per_min": 0.0, "rate_uM_per_s": 0.0, "series": "blank", "used_for_fit": false, "initial_rate_eligible": false, "blank_for_substrate_uM": 200.0}, "plate:D5": {"enzyme_volume_ul": 0.0, "tmb_volume_ul": 16.0, "buffer_volume_ul": 184.0, "total_volume_ul": 200.0, "substrate_uM": 400.0, "enzyme_concentration_nM": 0.0, "enzyme_concentration_ng_ml": 0.0, "enzyme_concentration_M": 0.0, "rate_A652_per_min": 0.0, "rate_uM_per_s": 0.0, "series": "blank", "used_for_fit": false, "initial_rate_eligible": false, "blank_for_substrate_uM": 400.0}}, "note": "Final well volumes and enzyme concentrations after the individual dispenses."}}
{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 164, "trace_sequence": 164, "timestamp": "2026-09-16T10:38:15+00:00", "logged_at": "2026-09-16T10:38:15.350000+00:00", "source_agent": "planner", "event_id": "msg-0164", "event_type": "recovery_decision", "rationale": "Reported KM = 100 uM. kcat = Vmax/[E] = 4011 /s at 10 ng/mL (0.2273 nM) HRP. Reported kcat uses the prepared-well concentration, 10 ng/mL. Vmax is 0.9116 uM/s, converted from 1.1945 A652/min with epsilon 39000 /M/cm and path 0.56 cm. That path is 0.28 cm times the well volume divided by 100 uL, which is 0.56 cm at 200 uL. The linear HRP working range is 0.5-10 ng/mL.", "message": "Reported KM = 100 uM. kcat = Vmax/[E] = 4011 /s at 10 ng/mL (0.2273 nM) HRP. Reported kcat uses the prepared-well concentration, 10 ng/mL. Vmax is 0.9116 uM/s, converted from 1.1945 A652/min with epsilon 39000 /M/cm and path 0.56 cm. That path is 0.28 cm times the well volume divided by 100 uL, which is 0.56 cm at 200 uL. The linear HRP working range is 0.5-10 ng/mL."}
{"record_kind": "event", "run_id": "pearl-manuscript-s5-20260916", "seq": 165, "trace_sequence": 165, "timestamp": "2026-09-16T10:38:27+00:00", "logged_at": "2026-09-16T10:38:27.350000+00:00", "source_agent": "planner", "event_id": "done-S5", "event_type": "completion_decision", "plan_id": "plan-s5", "revision_id": "rev_0", "adopted": {"summary": "Same-well characterization adopted.", "KM_uM": 100.0, "kcat_per_s": 4011.0, "Vmax_M_per_s": 9.115909090909091e-07, "Vmax_uM_per_s": 0.9115909090909091, "Vmax_A652_per_min": 1.1945487272727273, "enzyme_concentration_M": 2.2727272727272728e-10, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "basis": "kcat_per_s = Vmax_M_per_s / enzyme_concentration_M", "read_mode": "same_well", "acid_stop": false, "enzyme_stock": "3 nM nominal, 2000 uL", "working_range_analyte": "HRP", "linear_working_range_hrp_ng_ml": [0.5, 10.0], "linear_working_range_hrp_nM": [0.011363636363636364, 0.22727272727272727], "preparation_id": "prep-s5-001"}, "discarded": [], "message": "Same-well characterization adopted."}
{"record_kind": "trial", "run_id": "pearl-manuscript-s5-20260916", "seq": 166, "trace_sequence": 166, "timestamp": "2026-09-16T10:38:39+00:00", "logged_at": "2026-09-16T10:38:39.350000+00:00", "source_agent": null, "scenario_id": "S5", "termination": "completed", "not_wet_lab": false, "adopted": {"summary": "Same-well characterization adopted.", "KM_uM": 100.0, "kcat_per_s": 4011.0, "Vmax_M_per_s": 9.115909090909091e-07, "Vmax_uM_per_s": 0.9115909090909091, "Vmax_A652_per_min": 1.1945487272727273, "enzyme_concentration_M": 2.2727272727272728e-10, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "basis": "kcat_per_s = Vmax_M_per_s / enzyme_concentration_M", "read_mode": "same_well", "acid_stop": false, "enzyme_stock": "3 nM nominal, 2000 uL", "working_range_analyte": "HRP", "linear_working_range_hrp_ng_ml": [0.5, 10.0], "linear_working_range_hrp_nM": [0.011363636363636364, 0.22727272727272727], "preparation_id": "prep-s5-001"}, "discarded": [], "commentary": "Each substrate well received 15.1515 uL of the 3 nM stock in 200 uL. Each well was read at 3, 6, and 9 s after TMB was dispensed. Reported KM = 100 uM and kcat = Vmax/[E] = 4011 /s.", "analysis_report": {"KM_uM": 100.0, "kcat_per_s": 4011.0, "kcat_calculation": {"equation": "kcat_per_s = Vmax_M_per_s / enzyme_concentration_M", "absorbance_conversion": "Vmax_M_per_s = Vmax_A652_per_min / (epsilon_per_M_cm * path_cm * 60)", "concentration_conversion": "ng/mL = nM * hrp_mw_g_per_mol / 1000", "KM_uM": 100.0, "kcat_per_s": 4011.0, "physical_kcat_per_s": 4011.0, "replicate_delta_uM_per_s": 0.002787232971406401, "fit_KM_uM": 99.99999999999994, "fit_Vmax_uM_per_s": 0.9115909090909089, "fit_r2": 0.9998016021087216, "Vmax_M_per_s": 9.115909090909091e-07, "Vmax_uM_per_s": 0.9115909090909091, "Vmax_A652_per_min": 1.1945487272727273, "epsilon_per_M_cm": 39000.0, "path_cm": 0.56, "wavelength_nm": 652, "hrp_mw_g_per_mol": 44000.0, "enzyme_stock_nM": 3.0, "enzyme_stock_volume_ul": 2000.0, "enzyme_stock_ng_ml": 132.0, "enzyme_volume_ul": 15.151515151515152, "neat_enzyme_ul": 15.151515151515152, "enzyme_source_nM": 3.0, "enzyme_source_well": "A1", "enzyme_source_labware": "reservoir", "dilution_factor": 1, "total_volume_ul": 200.0, "enzyme_concentration_M": 2.2727272727272728e-10, "enzyme_concentration_nM": 0.22727272727272727, "enzyme_concentration_ng_ml": 10.0, "physical_enzyme_concentration_M": 2.2727272727272728e-10, "physical_enzyme_concentration_nM": 0.22727272727272727, "physical_enzyme_concentration_ng_ml": 10.0, "enzyme_concentration_ng_ml_from_label": 10.0, "kcat_basis": "prepared_well", "apparent_kcat_per_s_on_label": 4011.0, "kcat_basis_note": "Reported kcat divides Vmax by the prepared-well enzyme concentration. The labelled stock and the dispense volumes produce that concentration."}, "working_range": {"analyte": "HRP", "criterion": "initial rate proportional to final HRP concentration", "fixed_substrate_uM": 200.0, "linear_ng_ml": [0.5, 10.0], "linear_nM": [0.011363636363636364, 0.22727272727272727], "assay_enzyme_ng_ml": 10.0, "assay_enzyme_nM": 0.22727272727272727, "points": [{"hrp_ng_ml": 0.5, "hrp_nM": 0.011363636363636364, "enzyme_volume_ul": 1.5151515151515151, "neat_enzyme_ul": 0.7575757575757576, "hrp_source": "H12", "hrp_source_labware": "plate", "enzyme_source_nM": 1.5, "dilution_factor": 2, "tmb_volume_ul": 8.0, "buffer_volume_ul": 190.4848484848485, "total_volume_ul": 200.0, "substrate_uM": 200.0, "rate_A652_per_min": 0.039818290909090906, "rate_uM_per_s": 0.030386363636363628, "in_linear_range": true}, {"hrp_ng_ml": 1.0, "hrp_nM": 0.022727272727272728, "enzyme_volume_ul": 1.5151515151515151, "neat_enzyme_ul": 1.5151515151515151, "hrp_source": "A1", "hrp_source_labware": "reservoir", "enzyme_source_nM": 3.0, "dilution_factor": 1, "tmb_volume_ul": 8.0, "buffer_volume_ul": 190.4848484848485, "total_volume_ul": 200.0, "substrate_uM": 200.0, "rate_A652_per_min": 0.07963658181818181, "rate_uM_per_s": 0.060772727272727256, "in_linear_range": true}, {"hrp_ng_ml": 2.0, "hrp_nM": 0.045454545454545456, "enzyme_volume_ul": 3.0303030303030303, "neat_enzyme_ul": 3.0303030303030303, "hrp_source": "A1", "hrp_source_labware": "reservoir", "enzyme_source_nM": 3.0, "dilution_factor": 1, "tmb_volume_ul": 8.0, "buffer_volume_ul": 188.96969696969697, "total_volume_ul": 200.0, "substrate_uM": 200.0, "rate_A652_per_min": 0.15927316363636362, "rate_uM_per_s": 0.12154545454545451, "in_linear_range": true}, {"hrp_ng_ml": 5.0, "hrp_nM": 0.11363636363636363, "enzyme_volume_ul": 7.575757575757576, "neat_enzyme_ul": 7.575757575757576, "hrp_source": "A1", "hrp_source_labware": "reservoir", "enzyme_source_nM": 3.0, "dilution_factor": 1, "tmb_volume_ul": 8.0, "buffer_volume_ul": 184.42424242424244, "total_volume_ul": 200.0, "substrate_uM": 200.0, "rate_A652_per_min": 0.3981829090909091, "rate_uM_per_s": 0.3038636363636363, "in_linear_range": true}, {"hrp_ng_ml": 10.0, "hrp_nM": 0.22727272727272727, "enzyme_volume_ul": 15.151515151515152, "neat_enzyme_ul": 15.151515151515152, "hrp_source": "A1", "hrp_source_labware": "reservoir", "enzyme_source_nM": 3.0, "dilution_factor": 1, "tmb_volume_ul": 8.0, "buffer_volume_ul": 176.84848484848484, "total_volume_ul": 200.0, "substrate_uM": 200.0, "rate_A652_per_min": 0.7963658181818182, "rate_uM_per_s": 0.6077272727272726, "in_linear_range": true}, {"hrp_ng_ml": 20.0, "hrp_nM": 0.45454545454545453, "enzyme_volume_ul": 30.303030303030305, "neat_enzyme_ul": 30.303030303030305, "hrp_source": "A1", "hrp_source_labware": "reservoir", "enzyme_source_nM": 3.0, "dilution_factor": 1, "tmb_volume_ul": 8.0, "buffer_volume_ul": 161.6969696969697, "total_volume_ul": 200.0, "substrate_uM": 200.0, "rate_A652_per_min": 1.4334584727272726, "rate_uM_per_s": 1.0939090909090905, "in_linear_range": false}, {"hrp_ng_ml": 40.0, "hrp_nM": 0.9090909090909091, "enzyme_volume_ul": 60.60606060606061, "neat_enzyme_ul": 60.60606060606061, "hrp_source": "A1", "hrp_source_labware": "reservoir", "enzyme_source_nM": 3.0, "dilution_factor": 1, "tmb_volume_ul": 8.0, "buffer_volume_ul": 131.39393939393938, "total_volume_ul": 200.0, "substrate_uM": 200.0, "rate_A652_per_min": 2.389097454545454, "rate_uM_per_s": 1.8231818181818178, "in_linear_range": false}, {"hrp_ng_ml": 80.0, "hrp_nM": 1.8181818181818181, "enzyme_volume_ul": 121.21212121212122, "neat_enzyme_ul": 121.21212121212122, "hrp_source": "A1", "hrp_source_labware": "reservoir", "enzyme_source_nM": 3.0, "dilution_factor": 1, "tmb_volume_ul": 8.0, "buffer_volume_ul": 70.78787878787878, "total_volume_ul": 200.0, "substrate_uM": 200.0, "rate_A652_per_min": 3.5836461818181817, "rate_uM_per_s": 2.734772727272727, "in_linear_range": false}]}}}