Programming bulk enzyme heterojunctions for biosensor development with tetrahedral DNA framework

Protein-protein interactions are spatially regulated in living cells to realize high reaction efficiency, as seen in naturally existing electron-transfer chains. Nevertheless, arrangement of chemical/biochemical components at the artificial device interfaces does not possess the same level of control. Here we report a tetrahedral DNA framework-enabled bulk enzyme heterojunction (BEH) strategy to program the multi-enzyme catalytic cascade at the interface of electrochemical biosensors. The construction of interpenetrating network of BEH at the millimeter-scale electrode interface brings enzyme pairs within the critical coupling length (CCL) of ~10 nm, which in turn greatly improve the overall catalytic cascade efficiency by ~10-fold. We demonstrate the BEH generality with a range of enzyme pairs for electrochemically detecting clinically relevant molecular targets. As a proof of concept, a BEH-based sarcosine sensor enables single-step detection of the metabolic biomarker of sarcosine with ultrasensitivity, which hold the potential for precision diagnosis of early-stage prostate cancer. Tetrahedral DNA framework-enabled bulk enzyme heterojunctions have been used to program biosensor interfaces. Here, the authors use DNA tetrahedrons to tether enzymes of an enzymatic cascade to gold electrodes, hence raising them over the bulk solution, which led to improved kinetics and sensitivity.