"Cells-on-Beads": A novel immobilization approach for the construction of whole-cell amperometric biosensors

Microbial cells are attractive biorecognition elements for electrochemical biosensing applications. A desired configuration is the immobilization of the cells onto the transducer's surface. Here we propose the design and demonstrate the feasibility of a novel 'cells-on-beads' (COB) immobilization approach, providing simple, fast, low cost and reproducible method for the construction of viable whole-cell biochips. The proposed immobilization approach is based on controlled chemical modification of polyacrylamide porous beads resulting in positively charged microcarriers exhibiting strong adsorption capabilities to both cells and gold surfaces. As the cells are physically adsorbed to the outer surface of the beads with no further treatments, this method is particularly suited for systems integrating sensitive cells with the detection of electroactive products susceptible to diffusion limitations. Such functional beads can be stored at 4 °C for at least six months and deposited on the biochip on demand. The COB approach was demonstrated using Escherichia coli (E. coli) cells expressing an intracellular enzyme, cytochrome P450 BM3, and aniline as model substrate. The current signal was generated by the oxidation of the secreted enzymatic product p-aminophenol on electrode's surface at 100 mV vs Ag/AgCl. The electrochemical biochip yielded a high and clear signal within the range of tens of nanoamperes that was linearly correlated to the substrate concentration. The proposed method was characterized and optimized and its relative advantage over a suspended cells system was illustrated.