Towards Highly Sensitive Multi-Analyte Micro-Biosensor Arrays for In Vitro Sensing

Cells and tissues produce many micron-scale extracellular chemical gradients and fluxes due to metabolism and for communication [1, 2]. These gradients and fluxes have an important role in physiology and pathology. Highly sensitive multi-analyte micro-biosensor arrays would be useful tools for simultaneously measuring the gradients and fluxes of many molecules from cells cultures in vitro. To this end, we have investigated (1) a practical means to functionalize MEAs with two or more enzymes, (2) maximizing the effective surface area of Pt-black-modified biosensor, comparing it to other nanomaterials, (3) the nature of electron transfer between glucose oxidase and single-walled carbon nanotubes, and (4) the effect H2O2 phosphate buffer saline, optimal for measurement of H2O2 gradients, has on the morphology and viability of a model cell culture (human astrocytes). We have developed a direct-writing method to specifically functionalize individual microelectrodes in an MEA with 100-150-μm dots of glucose oxidase and lactate oxidase in cross-linked 15% PEGDA-600. We determined parameters for pulsed potentiostatic deposition of Pt black. With respect to electron transfer between glucose oxidase and carbon nanotubes, our data suggest that glucose oxidase spontaneously adsorbs to single-walled carbon nanotubes, and once it adsorbs, quasi-reversible one-electron transfer occurs near -0.5 V vs Ag/AgCl during cyclic voltammetry in pH 7.4 PBS. However, the glucose oxidase that adsorbs to the carbon nanotubes is no longer catalytically active. Finally, two-hour exposure to 20 -500 μMH2O2 in only pH 7.4 PBS at room temperature drastically changes human astrocyte morphology and adherance. However, most of the cells are still alive up to that time. These developments and findings have brought us closer to a highly sensitive multi-analyte micro-biosensor that can simultaneously measure glucose and lactate fluxes from cell cultures over hours. The enzyme functionalization technique may also be useful for making fully printed flexible multi-analyte sensors and micro-biofuel-cells