Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection

Abstract

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.

Keywords: arsenic resistance; positive feedback amplifier; sensitivity; specificity; whole-cell biosensor (WCB).

FIG 1

Schematic of the arsenic WCBs with positive feedback (B) and without (A). (A) The typical arsenic WCB consists of the ArsR-regulated promoter P_ars, the regulator arsR, and the reporter gene mCherry. (B) The positive feedback WCB consists of the arsR-P_ars regulatory circuit and a positive feedback amplifier where LuxR produced in response to arsenite activates the expression of mCherry and LuxR from the P_luxI promoter. The LuxR from the P_luxI promoter activates its own expression and forms a positive feedback loop.

FIG 2

Growth curves of the two WCBs at different concentrations of arsenite. (A) WCB without positive feedback; (B) WCB with positive feedback.

FIG 3

Time-dependent response of arsenic biosensors with (A) and without (B) positive feedback. Biosensor cells were grown for 10 h at 0 μM, 0.01 μM, 0.1 μM, and 10 μM As(III). Statistical significance was shown as follows: *, P < 0.01; **, P < 0.001.

FIG 4

Dose-response curves of arsenic biosensors with (red solid squares) and without (black solid squares) the positive feedback amplifier. Biosensors were grown for 9 h at different arsenite concentrations from 0 μM to 200 μM. The right y axis indicates that the maximum FIR value is set to 1. Statistical significance was shown as follows: *, P < 0.01; **, P < 0.001.

FIG 5

Arsenic specificity of the biosensors with and without the positive feedback amplifier. Fluorescence intensity of the biosensors was measured after exposure to various metals at concentrations of 0.01 μM, 0.1 μM, 1 µM, and 10 µM for 8 h.