Optimized enzyme matrices for 3D-printed microneedle biosensors

Microneedle (MN) technology offers an advanced approach for minimally invasive biosensing by providing non-painful access to interstitial fluid (ISF) and enabling continuous, real-time monitoring. The dermis (1-3 mm thick) is the skin layer where the ISF content is maximum (40 % of the water content is ISF)¹. Thus, MN structures must be in the range from 200 to 1500 µm to penetrate the outer skin layers and reach the ISF.

In this study, MN patches were designed and fabricated via 3D printing; then, MN were transformed into electrodes for electrochemical sensing by gold sputtering. Silver-filled vias were used for electrical interconnection to the potentiostat contacts and Prussian blue (PB) was used as an artificial peroxidase, being electrodeposited on the MN tips to facilitate the detection of glucose and lactate in the presence of their respective oxidase enzymes².

The primary objective of this study was to enhance the performance of these biosensors by optimizing the enzyme immobilization matrix, a crucial determinant of sensor stability and analytical efficiency. Three different biopolymer-based hydrogels—chitosan (CHI), silk fibroin (SF), and methacrylated hyaluronic acid (MeHA)—were evaluated for their ability to maintain enzymatic activity, minimize enzyme leaching, and modulate analyte transport. Diffusion behavior within these matrices was assessed using ferrocyanide as a model electroactive probe on gold microelectrodes. Crosslinking was employed in all hydrogel systems to reinforce structural integrity and promote enzyme retention. The results demonstrate that the choice of immobilization medium significantly affects both sensitivity and dynamic range, providing insights for the development of robust, wearable MN biosensors for applications such as diabetes management.