AbstractFusobacterium nucleatum has been correlated to many poor human conditions including oral infections, adverse pregnancies and cancer, and thus molecular tools capable of detecting this human pathogen can be used to develop diagnostic tests for them. Using a new selection method targeting thermally stable proteins without a counter‐selection step, we derived an fluorogenic RNA‐cleaving DNAzyme, named RFD‐FN1, that can be activated by a thermally stable protein target that is unique to F. nucleatum subspecies. High thermal stability of protein targets is a very desirable attribute for DNAzyme‐based biosensing directly with biological samples because nucleases found inherently in these samples can be heat‐inactivated. We further demonstrate that RFD‐FN1 can function as a fluorescent sensor in both human saliva and human stool samples. The discovery of RFD‐FN1 paired with a highly thermal stable protein target presents opportunities for developing simpler diagnostic tests for this important pathogen.

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