Protein Melting Biosensors for Label-Free and Self-Reporting of Iron-Binding Proteins and Iron Ion

Protein fluorescence sensing holds great promise for food safety, disease diagnosis, and environmental monitoring. Nevertheless, the synthesis of specific fluorescent dye probes is usually necessary, which significantly increases the cost and complexity of the synthesis. Herein, we developed a label-free, melting-based protein sensing strategy. The melting biosensors monitor the change in fluorescence intensity produced by aromatic fluorescent amino acids during protein unfolding and determine the melting temperature (<i>T</i>_m) of the protein. Based on that, we developed a Fe³⁺-associated <i>T</i>_m-regulated protein melting biosensor for rapid (detection step <3 min) and high-throughput (>48 samples/experiment) detection of various iron-binding proteins. The shift in the <i>T</i>_m value induced by the binding of proteins to Fe³⁺ and the peak height of Fe³⁺ proteins corresponding to varying protein concentrations can accurately enable the self-reporting detection. Conversely, iron-binding proteins can be used as detection probes, and this system can also be applied for Fe³⁺ detection. In addition, the introduction of melting biosensors can eliminate background fluorescence. The proposed protein melting biosensors enable label-free, rapid, and accurate detection of targets in complex biological samples, and they have the potential to be extended to the sensitive detection of different proteins (e.g., metal-binding proteins).