Versatile Protein-A Coated Photoelectric Immunosensors with a Purple-Membrane Monolayer Transducer Fabricated by Affinity-Immobilization on a Graphene-Oxide Complexed Linker and by Shear Flow.

Bacteriorhodopsin-embedded purple membranes (PM) have been demonstrated to be a sensitive photoelectric transducer for microbial detection. To efficiently prepare versatile BR-based immunosensors with protein A as antibody captures, a large, high-coverage, and uniformly oriented PM monolayer was fabricated on an electrode as an effective foundation for protein A conjugation through bis-NHS esters, by first affinity-coating biotinylated PM on an aminated surface using a complex of oxidized avidin and graphene oxide as the planar linker and then washing the coating with a shear flow. Three different polyclonal antibodies, each against Escherichia coli, Lactobacillus acidophilus, and Streptococcus mutans, respectively, were individually, effectively and readily adsorbed on the protein A coated electrodes, leading to selective and sensitive quantitative detection of their respective target cells in a single step without any labeling. A single-cell detection limit was achieved for the former two cells. AFM, photocurrent, and Raman analyses all displayed each fabricated layer as well as the captured bacteria, with AFM particularly revealing the formation of a massive continuous PM monolayer on aminated mica. The facile cell-membrane monolayer fabrication and membrane surface conjugation techniques disclosed in this study may be widely applied to the preparation of different biomembrane-based biosensors.

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 107CFU/10mL 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.

Effect of graphene oxide on affinity-immobilization of purple membranes on solid supports.

The effect of graphene oxide (GO) on the surface fabrication of purple membranes (PM) containing photosensitive bacteriorhodopsin is first reported in this study. GO was initially modified with biotin and then coupled with oxidized avidin to generate a GO-avidin complex, which was subsequently used as a linker to immobilize biotinylated PM (b-PM) onto amine-functionalized supports. Indium-tin-oxide glass coated with the GO-avidin complex was more hydrophilic than the electrode coated only with oxidized avidin, and the successive b-PM adsorption yielded a 1.4-fold higher (410 nA/cm(2)) photoelectric activity. AFM analysis on mica revealed that the GO-avidin complex layer had less surface roughness and dissipation energy than the pure oxidized avidin linker layer. For subsequent b-PM fabrication, GO addition not only reduced the stacking of immobilized b-PM patches but also improved their interior compactness and surface smoothness. This study demonstrates a convenient way to introduce GO into PM fabrication technology to provide enhanced surface morphology and photoelectric activity.