In this study, a cell-free electrochemical assay based on allosteric transcription factors (aTFs) and CRISPR-Cas14a was developed for the detection of progesterone in trace samples. This electrochemical biosensor helps to overcome the drawbacks of the traditional fluorescence assay based on the CRISPR-Cas system and aTFs combined for non-nucleic acid targets that is poorly effective for the detection of colored samples. By comparing and optimizing the concentration and length of the probes in the straight chain and hairpin structure, the sensor performance was improved. In addition, different sgRNA from other studies was designed to overcome the effect of sequence folding in the space region on Cas14a activation. Based on these optimization results, we constructed an electrochemical sensor for progesterone quantification in the range of 66.7pM to 3.33 × 10μM. This method requires only 2 μL of sample and does not necessitate complex pretreatment steps, with detection completed within 1.5 h. The method has been successfully applied to food, environmental, and biological samples, with recovery rates between 82.65% and 109%. This suggests that CRISPR and allosteric transcription factor-powered electrochemical detection methods have significant potential for use in the field of small molecule detection under various scenarios.
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