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Highly Sensitive Whole-Cell Biosensor for Cadmium Detection Based on a Negative Feedback Circuit.

ABSTRACT: Although many whole-cell biosensors (WCBs) for the detection of Cd²⁺ have been developed over the years, most lack sensitivity and specificity. In this paper, we developed a Cd²⁺ WCB with a negative feedback amplifier in P. putida KT2440. Based on the slope of the linear detection curve as a measure of sensitivity, WCB with negative feedback amplifier greatly increased the output signal of the reporter mCherry, resulting in 33% greater sensitivity than in an equivalent WCB without the negative feedback circuit. Moreover, WCB with negative feedback amplifier exhibited increased Cd²⁺ tolerance and a lower detection limit of 0.1 nM, a remarkable 400-fold improvement compared to the WCB without the negative feedback circuit, which is significantly below the World Health Organization standard of 27 nM (0.003 mg/L) for cadmium in drinking water. Due to the superior amplification of the output signal, WCB with negative feedback amplifier can provide a detectable signal in a much shorter time, and a fast response is highly preferable for real field applications. In addition, the WCB with negative feedback amplifier showed an unusually high specificity for Cd²⁺ compared to other metal ions, giving signals with other metals that were between 17.6 and 41.4 times weaker than with Cd²⁺. In summary, the negative feedback amplifier WCB designed in this work meets the requirements of Cd²⁺ detection with very high sensitivity and specificity, which also demonstrates that genetic negative feedback amplifiers are excellent tools for improving the performance of WCBs.

SUBMITTER: Zhang G

PROVIDER: S-EPMC8678453 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Whole-cell biosensors (WCBs) have been designed to detect As(III), but most suffer from poor sensitivity and specificity. In this paper, we developed an arsenic WCB with a positive feedback amplifier in Escherichia coli DH5α. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier. The sensitivity of the WCB with positive feedback is about 1 order of magnitude higher than that without positive feedback when evaluated using a half-saturation As(III) concentration. The minimum detection limit for As(III) was reduced by 1 order of magnitude to 0.1 µM, lower than the World Health Organization standard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 µM. Due to the amplification of the output signal, the WCB was able to give detectable signals within a shorter period, and a fast response is essential for in situ operations. Moreover, the WCB with the positive feedback amplifier showed exceptionally high specificity toward As(III) when compared with other metal ions. Collectively, the designed positive feedback amplifier WCB meets the requirements for As(III) detection with high sensitivity and specificity. This work also demonstrates the importance of genetic circuit engineering in designing WCBs, and the use of genetic positive feedback amplifiers is a good strategy to improve the performance of WCBs.IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simple methods for the sensitive and specific monitoring of arsenic concentration in drinking water are needed. In this study, we designed an arsenic WCB with a positive feedback amplifier. It is highly sensitive and able to detect arsenic below the WHO limit level. In addition, it also significantly improves the specificity of the biosensor toward arsenic, giving a signal that is about 10 to 20 times stronger in response to As(III) than to other metals. This work not only provides simple but effective arsenic biosensors but also demonstrates the importance of genetic engineering, particularly the use of positive feedback amplifiers, in designing WCBs.

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Highly Sensitive Whole-Cell Biosensor for Cadmium Detection Based on a Negative Feedback Circuit.

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