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The application of microbial fuel cell (MFC)-based toxicity sensors to real-world water monitoring is partly impeded by the limited sensitivity. To address this limitation, this study optimized the flow configurations and the control modes. Results revealed that the sensitivity increased by ∼15–41 times with the applying of a flow-through anode, compared to those with a flow-by anode. The sensors operated in the controlled anode potential (CP) mode delivered better sensitivity than those operated in the constant external resistance (ER) mode over a broad range of anode potentials from −0.41 V to +0.1 V. Electrodeposition of Cu(II) was found to bias the toxicity measurement at low anode potentials. The optimal anode potential was approximately −0.15 V, at which the sensor achieved an unbiased measurement of toxicity and the highest sensitivity. This value was greater than those required for electrodeposition while …