Replacing Manual Operation with Bio-Automation II: Construction of a Biological Digestion Gene Circuit to Eliminate the Interference of Food Matrices in the Rapid Detection of Heavy Metals.

Abstract

Food matrices such as phytic acid, starch, and proteins can chelate heavy metals, acting as stabilizers that significantly hinder accurately detecting heavy metal contamination. This study proposes a biological digestion strategy to overcome such interference. The gene sequences for phytase (<i>appA</i>) from <i>Escherichia coli (E. coli)</i>, α-amylase (<i>amyA</i>) from <i>Escherichia coli (E. coli)</i>, and protease (<i>AO090120000474</i>) from <i>Aspergillus oryzae</i> were identified via bioinformatics screening. Whole-cell biosensors were then developed to simultaneously detect mercury ions (Hg²⁺) and digest phytate, starch, and proteins. In the presence of 100 μM Hg²⁺, biosensor responses improved by 1.43-, 1.38-, and 1.11-fold, respectively. A "heavy metal pollutant bio-digestion pathway" was constructed by integrating genes for synthesizing phytic acid, starch, and protein with those for Hg²⁺ detection. In the presence of 100 μM Hg²⁺, the detection effect was improved by 1.36-fold. The detection limit of the BαAP whole-cell biosensor was 0.082 μM, while the limit of quantitation was 0.272 μM. The study effectively addresses the limitations of biosensor performance in real sample detection.