ABSTRACT
The microarray format has allowed for rapid and sensitive detection of thousands of analyte DNAs in a single sample, and there is considerable interest in extending this technology to protein biosensing. While glass is the most common substrate for microarrays, its binding capacity is limited because the glass surface is flat. One way to overcome this limitation is to develop arrays based on porous materials. Such "3-D" arrays can provide greater sensitivity because both the capture molecules and the analyte species they bind are immobilized throughout the thickness of the porous material. We describe here 3-D protein microarrays based on nanopore alumina membranes that contain silica nanotubes within the pores. These microarrays are prepared via a plasma-etch method using a TEM grid as the etch mask and consist of individual nanotube-containing microwells imbedded in a Ag film that coats the alumina membrane surface. We show that the microwells can be functionalized with antibodies and that these antibodies can capture their antigen proteins, which serve as prototype analytes. The analyte proteins are fluorescently tagged, which allows for fluorescence microscopy-based imaging of the array. The Ag surrounding the microwells shows very low background fluorescence, thus improving the signal-background ratio obtained from these arrays.
Subject(s)
Nanotubes/chemistry , Protein Array Analysis/methods , Proteins/chemistry , Silicon Dioxide/chemistry , Electrochemistry , Immunoglobulin G/immunology , Microscopy, Electron, Scanning , Nanotubes/ultrastructure , Spectrometry, FluorescenceABSTRACT
There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. The nanopore most often used is the alpha-hemolysin protein channel, and the sensor consists of a single channel embedded within a lipid bilayer membrane. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current associated with translocation of the analyte through the channel-stochastic sensing. While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biological nanopore with an abiotic equivalent. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mechanical and chemically robust polymeric membrane. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not observed. Instead, the protein analyte binds to a biochemical molecular-recognition agent at the mouth of the conical nanotube, resulting in complete blockage of the ion current. Three different molecular-recognition agents, and correspondingly three different protein analytes, were investigated: (i) biotin/streptavidin, (ii) protein-G/immunoglobulin, and (iii) an antibody to the protein ricin with ricin as the analyte.
Subject(s)
Biosensing Techniques/methods , Gold/chemistry , Nanotubes/chemistry , Proteins/chemistry , Animals , Antibodies/chemistry , Biotin/chemistry , Horses , Immunoglobulin G/analysis , Nerve Tissue Proteins/chemistry , Proteins/analysis , Ricin/analysis , Ricin/immunology , Streptavidin/chemistryABSTRACT
We have been investigating synthetic nanopore membranes that mimic the function of ligand-gated ion channels. We showed previously that the transmembrane ion current in a hydrophobic alumina nanopore membrane can be switched from an "off" state to an "on" state by exposure of the membrane to hydrophobic ionic surfactants. In these prior experiments, external electrodes and an external power supply were used to drive the ion current when the membrane was in its "on" state. In biological channels there are no electrodes, and the ion current is driven by an electrochemical potential difference across the cell membrane. In this article we mimic this function of the ligand-gated ion channel by applying a porous battery cathode film to one face of the hydrophobic alumina membrane and a porous battery anode film to the other face. Hence, in analogy to the naturally occurring channel case, we have a membrane with a built in electrochemical potential difference across the membrane. We show here that in the absence of the ligand (again, a hydrophobic ionic surfactant), the membrane is in its "off" state, and the electrochemical potential difference cannot be utilized to drive a transmembrane ion current. In contrast, when the ligand is detected, the membrane switches to its "on" state and the transmembrane battery discharges, producing a corresponding transmembrane ion current.
Subject(s)
Biomimetic Materials/chemistry , Electric Power Supplies , Ion Channels/chemistry , Membrane Potentials , Membranes, Artificial , Microelectrodes , Nanotechnology/methods , Surface-Active Agents/chemistry , Biomimetic Materials/chemical synthesis , Ion Channel Gating , Materials Testing , Nanotechnology/instrumentation , PorosityABSTRACT
Electroless deposition of gold on the pore walls of polycarbonate templates is currently the best known method for controlling inside diameters of template-synthesized nanotubes. It would be very useful to have alternative template-based synthetic chemistries that yield nanotubes composed of other materials, but which still allow for precise control over the nanotube wall thickness and i.d. A film-formation process that is based on layer-by-layer deposition of the film-forming material along the pore walls of the template membrane provides this desired alternative synthetic chemistry. We describe here the use of Mallouk's alpha,omega-diorganophosphonate/Zr layer-by-layer film-forming method for preparing nanotubes within the pores of alumina template membranes. We have found that this method allows accurate, quantitative, and predictable control over the wall thickness, and thus i.d., of the layered nanotubes obtained.
ABSTRACT
Tube-shaped nanostructures (nanotubes) have a number of attributes that make them potentially useful for biomedical applications such as drug delivery/detoxification and enzyme immobilization. Template synthesis provides a route for preparing monodisperse nanotubes of nearly any size and composed of nearly any material. We show here that template-synthesized silica nanotubes can be biochemically functionalized such that they act as biocatalysts and highly selective nano-phase extraction agents for bioseparations. For example, nanotubes containing an enantioselective antibody selectively extract the enantiomer of a drug molecule that binds to the antibody, relative to the enantiomer that has no specific interaction with the antibody. Nanotubes containing the enzyme glucose oxidase function as nanophase bioreactors to catalyze the oxidation of glucose.
Subject(s)
Nanotechnology/methods , Silicon Dioxide/chemistry , Antibodies/chemistry , Catalysis , Chromatography, High Pressure Liquid/methods , Fluorescent Dyes/chemistry , Silanes/chemistryABSTRACT
Synthetic bio-nanotube membranes were developed and used to separate two enantiomers of a chiral drug. These membranes are based on alumina films that have cylindrical pores with monodisperse nanoscopic diameters (for example, 20 nanometers). Silica nanotubes were chemically synthesized within the pores of these films, and an antibody that selectively binds one of the enantiomers of the drug was attached to the inner walls of the silica nanotubes. These membranes selectively transport the enantiomer that specifically binds to the antibody, relative to the enantiomer that has lower affinity for the antibody. The solvent dimethyl sulfoxide was used to tune the antibody binding affinity. The enantiomeric selectivity coefficient increases as the inside diameter of the silica nanotubes decreases.