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

Abstract

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 and Label-Free Determination of Human Glycated Hemoglobin Levels Using Bacteriorhodopsin as the Biosensor Transducer

“…ITO glass electrodes were coated with b-PM monolayers, which are termed b-PM chips, and were prepared as previously described [ 24 ]. APPA-aminated ITO glass was drop-coated with a GO-OA complex linker, which was prepared by mixing GO and OA at a 1:10 weight ratio, and then 1.5 mg/mL b-PM (coating diameter: 3 mm) and was subsequently washed with a laminar shear flow (Reynolds number = 0.9) inside a microfluidic setup.…”

“…This method of detection is verified using a BR-based Hb immunosensor that is prepared with a versatile protein A-conjugated PM electrode that was developed by the authors [ 24 ]. Two other aptasensors are devised by covalently attaching HbA0 and HbA1c-specific nucleic acid aptamers [ 18 ] on PM electrodes to, respectively, quantify HbA0 and HbA1c.…”

“…Bacteriorhodopsin (BR), which is a natural photosensor protein that resides in the purple membrane (PM) of Halobacterium salinarum, is the photoelectric transducer for novel immunosensors that have recently been developed for direct and label-free detection of microorganisms [ 23 , 24 ]. BR naturally exists as trimers that are arranged in hexagonal lattices of 6.2 ± 0.2 nm side length and which are repeatedly and densely assembled inside a stable 5-nm-thick two-dimensional (2D) PM with a packing density of around 2.6 × 10 4 trimers/μm 2 , according to the atomic force microscopy images (AFM) [ 25 ].…”

“…Driven by the photoisomerization of its retinal chromophore, each illuminated BR monomer pumps a proton uni-directionally from the cytoplasmic (CP) to the extracellular (EC) side of PM and produces a photoelectric effect that is applied in bioelectronics devices [ 26 , 27 ]. The photocurrent that is produced by BR correlates well with the incident light intensity [ 28 ] so the light-shielding bacterial cells that are captured by antibodies that are attached to PM-coated electrodes are readily detected using the reduction in photocurrent in the immunosensors as a simple quantification parameter [ 23 , 24 ]. Material that binds on the immobilized BR molecules may also cause restricted movement in the helices that are involved in proton transportation and contribute to the reduction in photocurrent generation during the layer-by-layer preparation of the sensor and in target binding [ 23 ].…”

“…Light-activated BR transports protons uni-directionally, so counter-oriented PM layers generate opposite proton fluxes and thus produce photocurrent ineffectively. To maximize sensor efficiency, the principle of strep(avidin)-biotin affinity adsorption is used to achieve unidirectional immobilization of PM layers on electrodes [ 23 , 24 ]. Moreover, double-sided avidin-linked graphene oxide (GO) sheets and shear flow are innovatively applied to PM fabrication to attain a high-coverage and uniformly oriented PM monolayer to effectively facilitate subsequent conjugation of recognition molecules on top [ 24 ].…”