A Facile Two-Step Enzymatic Approach for Conjugating Proteins to Polysaccharide Chitosan at an Electrode Interface

Biological components are integrated with electronic devices to create microsystems with novel functions and chitosan, a naturally occurring biopolymer, can play a significant role as an interface material. Chitosan can be electrodeposited within confined geometries by cathodic charge and appropriate electrode design and proteins can be conjugated to chitosan. However, conjugation chemistries can be slow and chitosan, a polycationic polysaccharide, enables non-specific binding in biofabrication processes. There is a need to speed up the assembly process and reduce non-specific binding. Here, we have developed a two-step methodology that accelerates protein assembly, reduces background and increases specificity. We first "coated" the surface of chitosan with a Lys-Tyr-Lys (KYK) tripeptide in a slow step using tyrosinase-mediated conjugation chemistry and then conjugated proteins with C-terminal glutamine-tags to the saturating KYK tripeptide via transglutaminase. As a demonstration, we assembled a functioning two-enzyme bacterial metabolic pathway on an electrode chip. Results indicated a fivefold decrease in non-specific binding and an improvement in signal to noise ratio from 0.3 to 20. This transglutaminase-mediated approach is simple and quick, it requires no chemical reagents, no printing or stamping devices; it employs biological components and is biologically benign to the component parts-all characteristics of biofabricated devices.