Whole-cell biosensors with regulatory circuits based on a synthetic dual-input promoter enabling the highly sensitive detection of ultra-trace Cd2+

The research on whole-cell biosensors tailored for trace and ultra-trace detection remains limited and the biosensors based on natural bacterial heavy metal resistance mechanisms have common issues of low sensitivity. In this study, we designed a promoter P _{<i>T7-cadO</i>} using the Cadmium ions (Cd²⁺)-binding protein binding site <i>cadO</i> with the T7 promoter at first and constructed a single-input whole-cell biosensor, which was named CP100, whose detection limit for Cd²⁺ met the WHO requirement, yet its response and sensitivity were quite low. We further introduced the <i>lacI</i> and <i>lac operator</i> (<i>lacO</i>) as the signal amplifier to construct a dual-input promoter P _{<i>T7-cadO-lacO-cadO</i>} and developed a biosensor named LC100, whose response and sensitivity were significantly improved, but background leakage became a new problem. Then we redesigned the gene circuit based on the regulatory circuit LCPM-2, which has the structure of "CadR-P _{<i>J23100</i>} -P _{<i>T7-cadO-lacO-cadO</i>} -mRFP1-LacI", with the LacI protein as the autoregulatory negative feedback model and finally obtained the biosensor named LC100-2, which achieved the detection of ultra-trace Cd²⁺ (0.00001-0.02 nM), with the sensitivity of 3748.22 times that of CP100. Moreover, LC100-2 demonstrated excellent specificity to Cd²⁺ among four other divalent metal ions and good anti-interference capability in the mixed divalent metal ions system. The results of the real water sample tests demonstrated that precise quantitative detection of Cd²⁺ with a final concentration of 0.001-0.02 nM could be achieved by adding only a small volume of the sample (1 μL). This finding showed promising application potential in the field of trace detection. Additionally, the regulatory circuit LCPM-2 based on the unique dual-input promoter enhanced responses and reduced background leakage simultaneously, thereby providing an innovative strategy for the design and simplification of biosensors' circuits.