Microfluidic device integrated with pre-reactor and dual enzyme-modified microelectrodes for monitoring in vivo glucose and lactate

A microfluidic device integrated with a micro-pre-reactor and glucose- and lactate-sensing microelectrodes in a dual channel was fabricated for in vivo monitoring using a micromachining technique. The device consists of two glass plates bonded together with UV-curable resin. The microelectrodes were made of carbon film, and a thin layer flow channel and a flow separator between the glucose and lactate electrodes were fabricated from 23-μm-thick positive photoresist. One carbon electrode was modified with a bilayer of glucose oxidase (GOD)/osmium-poly(vinylpyridine) wired horseradish peroxidase (Os-gel-HRP) and the other was modified with a bilayer of lactate oxidase (LOX)/Os-gel-HRP. The other glass plate has two rectangular flow channels created by chopper cutting with a dicing saw to enable a microdialysis (MD) probe and an outlet tube to be fitted. The cathodic currents of both the glucose and lactate electrodes started to increase rapidly after a microdialysis probe was dipped in glucose or lactate solution, and quickly reached a steady state value. We observed no chemical crosstalk between the glucose and lactate electrodes under our measurement conditions despite the short distance between them. By combining the device with a microdialysis probe, we obtained a linear relationship between the cathodic currents and glucose and lactate concentrations of 5 μM to 5 mM, which covers a concentration range sufficient for measuring brain glucose and lactate. The effect of l-ascorbic acid (l-AA), which is known to cause major interference during in vivo measurement, was eliminated when ascorbate oxidase was incorporated in the device upstream of the thin-layer flow channel. We successfully observed a glucose concentration decrease and lactate concentration increase in a rat brain when we perfused the striatum with veratridine solution.