Self-Sufficient and Autosensing Biocatalytic Inks for 3D-Printed Monolithic Bioreactors

Additive manufacturing, commonly known as three-dimensional (3D) printing, transforms simple in silico designs into real objects with accessibility, reproducibility, and precision. By merging the versatility of 3D printing with the inherent advantages of enzymatic processes, this technology opens up new possibilities for optimizing enzyme immobilization in continuous flow reactors. Here, we systematically investigate various formulations to develop an optimal biocatalytic ink capable of encapsulating enzymes and cofactors within a hydrogel matrix. The ink, composed of agarose and polyethylenimine (PEI), printed as porous monoliths, improved enzyme retention and cofactor absorption through ionic interactions, outperforming alternative formulations. By further integrating gold nanorods into the system, reaction substrates and intermediates (i.e., NAD⁺, isopropanol) can be detected through in operando surface enhanced Raman scattering (SERS) sensing, serving as a complementary tool for fluorescence microscopy. Using this optimized ink, we fabricated 3D-printed reactors with diverse architectures to evaluate their efficiency in the continuous flow reduction of ethyl acetoacetate. Reactors with a cross-shaped design exhibit stable product yields and minimize enzyme and cofactor leaching during continuous operation. Hence, we formulate and print a self-sufficient biocatalytic ink capable of sustaining the activity of immobilized dehydrogenases in continuous flow reactions without the addition of exogenous cofactors.