Lipid bilayer composition can influence the orientation of proteorhodopsin in artificial membranes.

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.

Label-free chemiresistive immunosensors for viruses.

We report development, characterization, and testing of chemiresistive immunosensors based on single polypyrrole (Ppy) nanowire for highly sensitive, specific, label free, and direct detection of viruses. Bacteriophages T7 and MS2 were used as safe models for viruses for demonstration. Ppy nanowires were electrochemically polymerized into alumina template, and single nanowire based devices were assembled on a pair of gold electrodes by ac dielectrophoretic alignment and anchored using maskless electrodeposition. Anti-T7 or anti-MS2 antibodies were immobilized on single Ppy nanowire using EDC-NHS chemistry to fabricate nanobiosensor for the detection of corresponding bacteriophage. The biosensors showed excellent sensitivity with a lower detection limit of 10(-3) plaque forming unit (PFU) in 10 mM phosphate buffer, wide dynamic range and excellent selectivity. The immunosensors were successfully applied for the detection of phages in spiked untreated urban runoff water samples. The results show the potential of these sensors in health care, environmental monitoring, food safety and homeland security for sensitive, specific, rapid, and affordable detection of bioagents/pathogens.

Single conducting polymer nanowire chemiresistive label-free immunosensor for cancer biomarker.

A simple and cost-effective, all-electrochemical method to fabricate and assemble single conducting polymer nanowire based biosensors was developed. Polypyrrole (Ppy) nanowires were synthesized by electrochemical polymerization using an alumina template. The single-nanowire chemoresistive sensor device was assembled using ac dielectrophoretic alignment followed by maskless anchoring on a pair of gold electrodes separated by 3 microm. To establish an efficient covalent surface biofunctionalization route, glutaraldehyde (GA) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) chemistries were compared. EDC was established to be the most effective chemistry and was used to surface-functionalize a single Ppy nanowire with cancer antigen (CA 125) antibody to fabricate a nanoimmunosensor for CA 125 biomarker detection and quantification. The immunosensor had excellent sensitivity with a lower detection limit of 1 U/mL CA 125 and dynamic range up to 1000 U/mL in 10 mM phosphate buffer. Furthermore, there was no loss of performance upon exposure to CA 125 in spiked human blood plasma. This demonstrates the clinical importance of these sensors for cancer marker detection with cost benefits and great portability for diagnosis of patients at the point of care.

Bioelectronic light-gated transistors with biologically tunable performance.

Light-activated bioelectronic silicon nanowire transistor devices are made by fusing proteoliposomes containing a bacteriorhodopsin (bR) proton pump onto the nanowire surface. Under green-light illumination, bR pumps protons toward the nanowire, and the pH gradient developed by the pump changes the transistor output. Furthermore, co-assembly of small biomolecules that alter the bilayer permeability to other ions can upregulate and downregulate the response of field-effect transistor devices.

Ordering in bio-inorganic hybrid nanomaterials probed by in situ scanning transmission X-ray microscopy.

Phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural variations that could critically impact the performance of the 1D phospholipid-Si NW composites. In this manuscript, we use scanning transmission X-ray microscopy (STXM) to probe bond orientation and bilayer thickness as a function of position with a spatial resolution of ∼30 nm for Δ9-cis 1,2-dioleoyl-sn-glycero-3-phosphocholine layers prepared Si NWs. When coupled with small angle X-ray scattering measurements, the STXM data reveal structural motifs of the Si NWs that give rise to multi-bilayer formation and enable assignment of the orientation of specific bonds known to affect the order and rigidity of phospholipid bilayers.

Effect of (L:D) Aspect Ratio on Single Polypyrrole Nanowire FET Device.

Effect of different aspect ratio (length to diameter ratio, L:D) on single polypyrrole (Ppy) nanowire based field effect transistor (FET) sensor for real time pH monitoring was studied. Ppy nanowires with diameters of ~60, ~80 and ~200 nm were synthesized using electrochemical deposition inside anodized aluminium oxide (AAO) template and were assembled using AC dielectrophoretic alignment followed by maskless anchoring on a pair of gold electrodes separated with different gap lengths. Microfabricated gold electrode patterns with gap size between 1 - 4 µm were developed by means of MEMS technique (photolithography). Using field effect transistor geometry with pair of microfabricated gold contact electrodes serving as a source and a drain, and a platinum (Pt) mesh (anchored in a microfluidic channel) was used as a gate electrode. When effect of different aspect ratio of the nanowire were compared, higher sensitivity was recorded for higher aspect ratio. The sensitivity was further improved by modulating the gate potential. These FET sensors based on single polypyrrole nanowire exhibited excellent and tunable sensitivity towards pH variations.

Conducting polymer nanowire-based chemiresistive biosensor for the detection of bacterial spores.

Polypyrrole nanowires (Ppy) were assembled onto microfabricated gold interdigitated microelectrodes, to construct a chemiresistive biosensor for the detection of Bacillus globigii, used as simulant of the threatening bioterrorism agent B. anthracis. The fabricated biosensor showed good linear correlation (r(2)=0.992) for low spore concentrations ranging from 1 to 100 CFU (colony forming units)/mL, a concentration that could be used in a bioterrorism attack, with a response time of 30 min, after which the sensor was saturated. The performance of the biosensor was also assessed in the absence of anti-B. globigii antibodies and in the presence of non-target bacterial cells (Escherichia coli) showing no significant non-specific interactions. We believe that Ppy nanowires are a good platform for the detection and also quantification of large molecules and biocomponents even at low concentrations.

Bioaffinity sensing using biologically functionalized conducting-polymer nanowire.

A simple, one-step method for fabricating single biologically functionalized conducting-polymer (polypyrrole) nanowire on prepatterned electrodes and its application to biosensing was demonstrated. The biologically functionalized polypyrrole was formed by the electropolymerization of an aqueous solution of pyrrole monomer and the model biomolecule, avidin- or streptavidin-conjugated ZnSe/CdSe quantum dots, within 100 or 200 nm wide by 3 mum long channels between gold electrodes on prefabricated silicon substrate. When challenged with biotin-DNA, the avidin- and streptavidin-polypyrrole nanowires generated a rapid change in resistance to as low as 1 nM, demonstrating the utility of the biomolecule-functionalized nanowire as biosensor. The method offers advantages of direct incorporation of functional biological molecules into the conducting-polymer nanowire during its synthesis, site-specific positioning, built-in electrical contacts, and scalability to high-density nanoarrays over the reported silicon nanowire and carbon nanotube biosensors.