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Single ion channel sensor Consider the problem: One obvious target for candidate drugs is single ion channel proteins, which form tunnels that control the passage of ions across cells or vesicle membranes. Electrochemistry is the conventional method for determining whether these tunnels are open or closed. However, studying the electrochemical properties of single ion channel proteins requires reducing the electrical background noise to only 10213–10212 A, which is achieved by electrically insulating the proteins from the surrounding environment (R > 109 V). Because traditional methods for doing this, such as patch clamp, are not suitable for automated high-throughput screening techniques, Christian Schmidt, Michael Mayer, and Horst Vogel of the Swiss Federal Institute of Technology developed a new chip-based approach using an “electrophoretic trap”. The trap consists of a thin insulating Si3N4 diaphragm with an aperture measuring between 0.6 and 7 µm. The Si3N4 is deposited on a silicon wafer, which has a microliter-sized fluid compartment etched into it. Because cells and native membrane vesicles are charged, an inhomogeneous electric field around the aperture is used to snare them from the solution and into the trap. To hold the snared cell or vesicle tightly to the trap, a 2-nm coating of SiO2 on the Si3N4 membrane is chemically modified to have a charged surface. This binds the captured cell or vesicle by electrostatic attraction. Fluid compartments on either side of the diaphragm contain low-impedance redox electrodes, which provide the electrical measurements. The researchers found that vesicles with diameters at least three times the aperture size were held tight against the aperture and did not wiggle through. The tight binding against the diaphragm provides the requisite high electrical insulation. These membranes were usually stable for >60 min. Experiments indicated that negatively charged vesicles, which were tightly
Vc PDMS SiO2 Si3N4 SiO2
Ag/AgCl
The trap in cross section, sandwiched between two layers of polydimethylsiloxane (PDMS) pads, and using Ag/AgCl redox electrodes for electrophoretic positioning and voltage recordings. (Adapted with permission. Copyright 2000 Wiley-VCH Verlag.)
bound to the surface, were flattened into planar unilamellar bilayers. Finally, the researchers succeeded in adding the self-integrating channel-forming protein
alamethicin to the vesicle and observed typical voltage-dependent activation. (Angew. Chem. Int. Ed. 2000, 39, 3137–3140)
Flow cytometry-based biosensor Multiple binding to proteins can be de-
based biosensor system for the detection
tected using distance-dependent fluo-
of cholera toxin (CT). Flow cytometry de-
rescence resonant energy transfer and
tected simultaneous double-fluorescence
fluorescence self-quenching. Biosensors
changes caused by multiple interactions
based on this approach have traditionally
between CT and the fluorophore-labeled
been used in conjunction with a spectro-
ganglioside GM1 on supported bilayers of
fluorometer. Xuedong Song, Basil Swan-
phospholipids.
son, and Jeane Shi of Los Alamos National
When used in conjunction with flow
Laboratory now demonstrate that biosen-
cytometry, the microsphere-based bio-
sors can be easily adapted to flow cytom-
sensor is capable of detecting 10 pM CT
etry, which offers enhanced sensitivity.
in 30 min. In addition, multiple targets
To accomplish this, the biosensor systems
can be simultaneously identified for high-
are simply constructed on the surfaces of
throughput applications, and free and
microspheres.
bound ligands can be continuously dis-
To demonstrate the technique, the researchers constructed a microsphere-
criminated for kinetic studies. (Anal. Biochem. 2000, 284, 35–41)
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