Electrocatalysis offers a means of electrochemical signal amplification, yet in DNA-based sensors, electrocatalysis has required high-density DNA films and strict assembly and passivation conditions. Here, we describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low-density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low-density DNA films. Electrocatalysis with methylene blue that is covalently tethered to the DNA by a flexible alkyl chain linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of a single cytosine-adenine (CA) mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: It is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling
