Direct, label-free, selective, and sensitive microbial detection using a bacteriorhodopsin-based photoelectric immunosensor

A photoelectric immunosensor using purple membranes (PM) as the transducer, which contains photoactive bacteriorhodopsin, is here first demonstrated for direct and label-free microbial detection. Biotinylated polyclonal antibodies against Escherichia coli were immobilized on a PM-coated electrode through further surface biotinylation and bridging avidin or NeutrAvidin. The photocurrent generated by the antibody-coated sensor was reduced after incubation with E. coli K-12 cultures, with the reduction level increased with the culture populations. The immunosensor prepared via NeutrAvidin exhibited much better selectivity than the one prepared via avidin, recognizing almost none of the tested Gram-positive bacteria. Cultures with populations ranging from 1 to 107 CFU/10 mL were detected in a single step without any preprocessing. Both AFM and Raman analysis confirmed the layer-by-layer fabrication of the antibody-coated substrates as well as the binding of microorganisms. By investigating the effect of illumination orientation and simulating the photocurrent responses with an equivalent circuit model containing a chemical capacitance, we suggest that the photocurrent reduction was primarily caused by the light-shielding effect of the captured bacteria. Using the current fabrication technique, versatile bacteriorhodopsin-based photoelectric immunosensors can be readily prepared to detect a wide variety of biological cells.